bims-proteo Biomed News
on Proteostasis
Issue of 2021–08–15
twenty-six papers selected by
Eric Chevet, INSERM
  1. TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins.
  2. Lipid droplets form a network interconnected by the endoplasmic reticulum through which their proteins equilibrate.
  3. TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.
  4. p62-containing, proteolytically active nuclear condensates, increase the efficiency of the ubiquitin-proteasome system.
  5. SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis.
  6. Structural and molecular bases to IRE1 activity modulation.
  7. Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila.
  8. Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage.
  9. The special unfolded protein response in plasma cells.
  10. Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex-dependent regulation of glycolysis.
  11. BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters.
  12. Hsf1 promotes hematopoietic stem cell fitness and proteostasis in response to ex vivo culture stress and aging.
  13. The Bacterial Hsp90 Chaperone: Cellular Functions and Mechanism of Action.
  14. N-Glycosylation Enhances Conformational Flexibility of Protein Disulfide Isomerase Revealed by Microsecond Molecular Dynamics and Markov State Modeling.
  15. Raising cGMP restores proteasome function and myelination in mice with a proteotoxic neuropathy.
  16. A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1.
  17. The structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client maturation state.
  18. Atypic SUMOylation of Nor1/NR4A3 regulates neural cell viability and redox sensitivity.
  19. Dual roles for LUBAC signaling in thymic epithelial cell development and survival.
  20. Hexokinases inhibit death receptor-dependent apoptosis on the mitochondria.
  21. Chemical methods for protein site-specific ubiquitination.
  22. ATF6 is required for efficient rhodopsin clearance and retinal homeostasis in the P23H rho retinitis pigmentosa mouse model.
  23. N-glycosylation patterns correlate with hepatocellular carcinoma genetic subtypes.
  24. Engineered protein-small molecule conjugates empower selective enzyme inhibition.
  25. Structural and biochemical advances on the recruitment of the autophagy-initiating ULK and TBK1 complexes by autophagy receptor NDP52.
  26. Hsp40s play complementary roles in the prevention of tau amyloid formation.
  1. Autophagy. 2021 Aug 12. 1-16
    TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins.
    Lin Lyu, Zheng Chen, Nami McCarty.
      Until recently, the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy were considered to be two independent systems that target proteins for degradation by proteasomes or via lysosomes, respectively. Here, we report that TRIM44 (tripartite motif containing 44) is a novel link that connects the UPS system with the autophagy degradation pathway. Suppressing the UPS degradation pathway leads to TRIM44 upregulation, which further promotes aggregated protein clearance through the binding of K48 ubiquitin chains on proteins. TRIM44 expression activates autophagy via promoting SQSTM1/p62 oligomerization, which rapidly increases the rate of aggregate protein removal. Overall, our data reveal that TRIM44 is a newly identified link between the UPS system and the autophagy pathway. Delineating the cross-talk between these two degradation pathways may reveal new mechanisms of targeting aggregate-prone diseases, such as cancer and neurodegenerative disease.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG5: autophagy related 5; BB: B-box domain; BECN1: beclin1; BM: bone marrow; CC: coiled-coil domain; CFTR: cystic fibrosis transmembrane conductance regulator; CON: control; CQ: chloroquine; DOX: doxycycline; DSP: dithiobis(succinimidly propionate); ER: endoplasmic reticulum; FI: fluorescence intensity; FL: full length; HIF1A/HIF-1#x3B1;: hypoxia inducible factor 1 subunit alpha; HSC: hematopoietic stem cells; HTT: huntingtin; KD: knockdown; KD-CON: knockdown construct control; MM: multiple myeloma; MTOR: mechanistic target of rapamycin kinase; NP-40: nonidet P-40; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; OE: overexpression; OE-CON: overexpression construct control; PARP: poly (ADP-ribose) polymerase; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; Tet-on: tetracycline; TRIM44: tripartite motif containing 44; UPS: ubiquitin-proteasome system; ZF: zinc-finger.
    Keywords:  Aggregates; TRIM44; autophagy; deubiquitinase; misfolded proteins; protein homeostasis; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1080/15548627.2021.1956105
  2. J Cell Sci. 2022 Mar 01. pii: jcs258819. [Epub ahead of print]135(5):
    Lipid droplets form a network interconnected by the endoplasmic reticulum through which their proteins equilibrate.
    Stéphanie Cottier, Roger Schneiter.
      Lipid droplets (LDs) are globular intracellular structures dedicated to the storage of neutral lipids. They are closely associated with the endoplasmic reticulum (ER) and are delineated by a monolayer of phospholipids that is continuous with the cytoplasmic leaflet of the ER membrane. LDs contain a specific set of proteins, but how these proteins are targeted to the LD surface is not fully understood. Here, we devised a yeast mating-based microscopic readout to monitor the transfer of LD proteins upon zygote formation. The results of this analysis indicate that ER fusion between mating partners is required for transfer of LD proteins and that this transfer is continuous, bidirectional and affects most LDs simultaneously. These observations suggest that LDs do not fuse upon mating of yeast cells, but that they form a network that is interconnected through the ER membrane. Consistent with this, ER-localized LD proteins rapidly move onto LDs of a mating partner and this protein transfer is affected by seipin, a protein important for proper LD biogenesis and the functional connection of LDs with the ER membrane.
    Keywords:   Saccharomyces cerevisiae ; Endoplasmic reticulum; Lipid droplets; Mating; Membrane fusion; Protein targeting; Seipin; Steryl esters; Triacylglycerol
    DOI:  https://doi.org/10.1242/jcs.258819
  3. Nat Commun. 2021 08 09. 12(1): 4794
    TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.
    Ying Qin, Qi Li, Wenbo Liang, Rongzhen Yan, Li Tong, Mutian Jia, Chunyuan Zhao, Wei Zhao.
      The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.
    DOI:  https://doi.org/10.1038/s41467-021-25033-4
  4. Proc Natl Acad Sci U S A. 2021 Aug 17. pii: e2107321118. [Epub ahead of print]118(33):
    p62-containing, proteolytically active nuclear condensates, increase the efficiency of the ubiquitin-proteasome system.
    Afu Fu, Victoria Cohen-Kaplan, Noa Avni, Ido Livneh, Aaron Ciechanover.
      Degradation of a protein by the ubiquitin-proteasome system (UPS) is a multistep process catalyzed by sequential reactions. Initially, ubiquitin is conjugated to the substrate in a process mediated by concerted activity of three enzymes; the last of them-a ubiquitin ligase (E3)-belongs to a family of several hundred members, each recognizing a few specific substrates. This is followed by repeated addition of ubiquitin moieties to the previously conjugated one to generate a ubiquitin chain that serves as a recognition element for the proteasome, which then degrades the substrate. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs). It stands to reason that efficiency of such a complex process would depend on colocalization of the different components in an assembly that allows the reactions to be carried out sequentially and processively. Here we describe nuclear condensates that are dynamic in their composition. They contain p62 as an essential component. These assemblies are generated by liquid-liquid phase separation (LLPS) and also contain ubiquitinated targets, 26S proteasome, the three conjugating enzymes, and DUBs. Under basal conditions, they serve as efficient centers for proteolysis of nuclear proteins (e.g., c-Myc) and unassembled subunits of the proteasome, suggesting they are involved in cellular protein quality control. Supporting this notion is the finding that such foci are also involved in degradation of misfolded proteins induced by heat and oxidative stresses, following recruitment of heat shock proteins and their associated ubiquitin ligase CHIP.
    Keywords:  LLPS condensates; p62; proteasome; protein degradation; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2107321118
  5. EMBO J. 2021 Aug 09. e107735
    SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis.
    Jörg Schweiggert, Gregor Habeck, Sandra Hess, Felix Mikus, Roman Beloshistov, Klaus Meese, Shoji Hata, Klaus-Peter Knobeloch, Frauke Melchior.
      Microtubule depolymerases of the kinesin-13 family play important roles in various cellular processes and are frequently overexpressed in different cancer types. Despite the importance of their correct abundance, remarkably little is known about how their levels are regulated in cells. Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5 , including the kinesin-13 member Kif2c/MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2a and Kif2b become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5  targets MCAK for proteasomal degradation predominantly during G2 . While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies and impairs ciliogenesis in the following G1 /G0 , which can be rescued by concomitant knockdown of MCAK, Kif2a or Kif2b. We thus propose a novel regulatory event of ciliogenesis that begins already within the G2 phase of the preceding cell cycle.
    Keywords:  Cullin-RING ligase; Fbxw5; MCAK; cilia; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2021107735
  6. Biochem J. 2021 Aug 13. 478(15): 2953-2975
    Structural and molecular bases to IRE1 activity modulation.
    Timothy Langlais, Diana Pelizzari-Raymundo, Sayyed Jalil Mahdizadeh, Nicolas Gouault, Francois Carreaux, Eric Chevet, Leif A Eriksson, Xavier Guillory.
      The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules.
    Keywords:  ER stress; IRE1; structure activity relationship (SAR); structure-based drug design (SBDD); unfolded protein response
    DOI:  https://doi.org/10.1042/BCJ20200919
  7. iScience. 2021 Aug 20. 24(8): 102819
    Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila.
    Peng Zhao, Ping Huang, Tongfu Xu, Xiaoxiang Xiang, Ying Sun, Jingqi Liu, Cheng Yan, Lei Wang, Jiamei Gao, Shang Cui, Xiangdong Wang, Lixing Zhan, Haiyun Song, Jingnan Liu, Wei Song, Yong Liu.
      The endoplasmic reticulum (ER)-resident transmembrane protein kinase/RNase Ire1 is a conserved sensor of the cellular unfolded protein response and has been implicated in lipid homeostasis, including lipid synthesis and transport, across species. Here we report a novel catabolic role of Ire1 in regulating lipid mobilization in Drosophila. We found that Ire1 is activated by nutrient deprivation, and, importantly, fat body-specific Ire1 deficiency leads to increased lipid mobilization and sensitizes flies to starvation, whereas fat body Ire1 overexpression results in the opposite phenotypes. Genetic interaction and biochemical analyses revealed that Ire1 regulates lipid mobilization by promoting Xbp1s-associated FoxO degradation and suppressing FoxO-dependent lipolytic programs. Our results demonstrate that Ire1 is a catabolic sensor and acts through the Xbp1s-FoxO axis to hamper the lipolytic response during chronic food deprivation. These findings offer new insights into the conserved Ire1 regulation of lipid homeostasis.
    Keywords:  Cell biology; Lipid; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.102819
  8. Mol Cell. 2021 Aug 06. pii: S1097-2765(21)00600-6. [Epub ahead of print]
    Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage.
    A Manuel Liaci, Barbara Steigenberger, Paulo Cesar Telles de Souza, Sem Tamara, Mariska Gröllers-Mulderij, Patrick Ogrissek, Siewert J Marrink, Richard A Scheltema, Friedrich Förster.
      The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.
    Keywords:  ER translocon; crosslinking mass spectrometry; cryo-EM; membrane thinning; molecular dynamics simulations; protein maturation; protein secretion; secretory pathway; signal peptidase complex; signal peptide
    DOI:  https://doi.org/10.1016/j.molcel.2021.07.031
  9. Immunol Rev. 2021 Aug 08.
    The special unfolded protein response in plasma cells.
    Daniela Ricci, Tali Gidalevitz, Yair Argon.
      The high rate of antibody production places considerable metabolic and folding stress on plasma cells (PC). Not surprisingly, they rely on the unfolded protein response (UPR), a universal signaling, and transcriptional network that monitors the health of the secretory pathway and mounts cellular responses to stress. Typically, the UPR utilizes three distinct stress sensors in the ER membrane, each regulating a subset of targets to re-establish homeostasis. PC use a specialized UPR scheme-they preemptively trigger the UPR via developmental signals and suppress two of the sensors, PERK and ATF6, relying on IRE1 alone. The specialized PC UPR program is tuned to the specific needs at every stage of development-from early biogenesis of secretory apparatus, to massive immunoglobulin expression later. Furthermore, the UPR in PC integrates with other pathways essential in a highly secretory cell-mTOR pathway that ensures efficient synthesis, autophagosomes that recycle components of the synthetic machinery, and apoptotic signaling that controls cell fate in the face of excessive folding stress. This specialized PC program is not shared with other secretory cells, for reasons yet to be defined. In this review, we give a perspective into how and why PC need such a unique UPR program.
    Keywords:  anticipatory unfolded protein response; autophagy; expansion of secretory apparatus; inactivation of sensors
    DOI:  https://doi.org/10.1111/imr.13012
  10. FASEB J. 2021 Sep;35(9): e21825
    Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex-dependent regulation of glycolysis.
    Matthew E R Maitland, Miljan Kuljanin, Xu Wang, Gilles A Lajoie, Caroline Schild-Poulter.
      Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.
    Keywords:  CTLH complex; LDHA; PKM; glycolysis; ubiquitination
    DOI:  https://doi.org/10.1096/fj.202100664R
  11. EMBO Rep. 2021 Aug 09. e51136
    BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters.
    Ashari R Kannangara, Daniel M Poole, Colten M McEwan, Joshua C Youngs, Vajira K Weerasekara, Alex M Thornock, Misael T Lazaro, Eranga R Balasooriya, Laura M Oh, Erik J Soderblom, Jonathan J Lee, Daniel L Simmons, Joshua L Andersen.
      ATG9A, the only multi-pass transmembrane protein among core ATG proteins, is an essential regulator of autophagy, yet its regulatory mechanisms and network of interactions are poorly understood. Through quantitative BioID proteomics, we identify a network of ATG9A interactions that includes members of the ULK1 complex and regulators of membrane fusion and vesicle trafficking, including the TRAPP, EARP, GARP, exocyst, AP-1, and AP-4 complexes. These interactions mark pathways of ATG9A trafficking through ER, Golgi, and endosomal systems. In exploring these data, we find that ATG9A interacts with components of the ULK1 complex, particularly ATG13 and ATG101. Using knockout/reconstitution and split-mVenus approaches to capture the ATG13-ATG101 dimer, we find that ATG9A interacts with ATG13-ATG101 independently of ULK1. Deletion of ATG13 or ATG101 causes a shift in ATG9A distribution, resulting in an aberrant accumulation of ATG9A at stalled clusters of p62/SQSTM1 and ubiquitin, which can be rescued by an ULK1 binding-deficient mutant of ATG13. Together, these data reveal ATG9A interactions in vesicle-trafficking and autophagy pathways, including a role for an ULK1-independent ATG13 complex in regulating ATG9A.
    Keywords:  ATG13; ATG9A; BioID; autophagy; p62
    DOI:  https://doi.org/10.15252/embr.202051136
  12. Cell Stem Cell. 2021 Aug 06. pii: S1934-5909(21)00294-0. [Epub ahead of print]
    Hsf1 promotes hematopoietic stem cell fitness and proteostasis in response to ex vivo culture stress and aging.
    Miriama Kruta, Mary Jean Sunshine, Bernadette A Chua, Yunpeng Fu, Ashu Chawla, Christopher H Dillingham, Lorena Hidalgo San Jose, Bijou De Jong, Fanny J Zhou, Robert A J Signer.
      Maintaining proteostasis is key to resisting stress and promoting healthy aging. Proteostasis is necessary to preserve stem cell function, but little is known about the mechanisms that regulate proteostasis during stress in stem cells, and whether disruptions of proteostasis contribute to stem cell aging is largely unexplored. We determined that ex-vivo-cultured mouse and human hematopoietic stem cells (HSCs) rapidly increase protein synthesis. This challenge to HSC proteostasis was associated with nuclear accumulation of Hsf1, and deletion of Hsf1 impaired HSC maintenance ex vivo. Strikingly, supplementing cultures with small molecules that enhance Hsf1 activation partially suppressed protein synthesis, rebalanced proteostasis, and supported retention of HSC serial reconstituting activity. Although Hsf1 was dispensable for young adult HSCs in vivo, Hsf1 deficiency increased protein synthesis and impaired the reconstituting activity of middle-aged HSCs. Hsf1 thus promotes proteostasis and the regenerative activity of HSCs in response to culture stress and aging.
    Keywords:  Hsf1; aging; heat shock response; hematopoiesis; hematopoietic stem cell; protein synthesis; proteostasis; stem cell; stress; translation
    DOI:  https://doi.org/10.1016/j.stem.2021.07.009
  13. Annu Rev Microbiol. 2021 Aug 10.
    The Bacterial Hsp90 Chaperone: Cellular Functions and Mechanism of Action.
    Sue Wickner, Thu-Lan Lily Nguyen, Olivier Genest.
      Heat shock protein 90 (Hsp90) is a molecular chaperone that folds and remodels proteins, thereby regulating the activity of numerous substrate proteins. Hsp90 is widely conserved across species and is essential in all eukaryotes and in some bacteria under stress conditions. To facilitate protein remodeling, bacterial Hsp90 collaborates with the Hsp70 molecular chaperone and its cochaperones. In contrast, the mechanism of protein remodeling performed by eukaryotic Hsp90 is more complex, involving more than 20 Hsp90 cochaperones in addition to Hsp70 and its cochaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of bacterial Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70. We describe the universally conserved structure and conformational dynamics of these chaperones and their interactions with one another and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide a framework for understanding the more complex eukaryotic Hsp90 system. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-micro-032421-035644
  14. J Phys Chem B. 2021 Aug 11.
    N-Glycosylation Enhances Conformational Flexibility of Protein Disulfide Isomerase Revealed by Microsecond Molecular Dynamics and Markov State Modeling.
    R Gregor Weiß, Marie-Estelle Losfeld, Markus Aebi, Sereina Riniker.
      Secreted proteins of eukaryotes are decorated with branched carbohydrate oligomers called glycans. This fact is only starting to be considered for in silico investigations of protein dynamics. Using all-atom molecular dynamics (MD) simulations and Markov state modeling (MSM), we unveil the influence of glycans on the conformational flexibility of the multidomain protein disulfide isomerase (PDI), which is a ubiquitous chaperone in the endoplasmic reticulum (ER). Yeast PDI (yPDI) from Saccharomyces cerevisiae is glycosylated at asparagine side chains and the knowledge of its five modified sites enables a realistic computational modeling. We compare simulations of glycosylated and unglycosylated yPDI and find that the presence of glycan-glycan and glycan-protein interactions influences the flexibility of PDI in different ways. For example, glycosylation reduces interdomain interactions, shifting the conformational ensemble toward more open, extended structures. In addition, we compare our results on yPDI with structural information of homologous proteins such as human PDI (hPDI), which is natively unglycosylated. Interestingly, hPDI lacks a surface recess that is present in yPDI. We find that glycosylation of yPDI facilitates its catalytic site to reach close to this surface recess. Hence, this might point to a possible functional relevance of glycosylation in yeast to act on substrates, while glycosylation seems redundant for the human homologous protein. We conclude that glycosylation is fundamental for protein dynamics, making it a necessity for a truthful representation of the flexibility and function in in silico studies of glycoproteins.
    DOI:  https://doi.org/10.1021/acs.jpcb.1c04279
  15. Brain. 2021 Aug 11. pii: awab249. [Epub ahead of print]
    Raising cGMP restores proteasome function and myelination in mice with a proteotoxic neuropathy.
    Jordan J S VerPlank, Joseph Gawron, Nicholas J Silvestri, M Laura Feltri, Lawrence Wrabetz, Alfred L Goldberg.
      Agents that raise cGMP by activating Protein Kinase G increase 26S proteasome activities, protein ubiquitination, and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot Marie Tooth 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate, and the Unfolded Protein Response (p-eif2 α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, but not of MPZWT. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil, to raise cGMP increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP, and p-elF2α. Furthermore, sildenafil treatment reduced the number of amyelinated axons, and increased myelin thickness and nerve conduction velocity in sciatic nerves. Thus, agents that raise cGMP, including ones widely used in medicine, may be useful therapies for CMT1B and other proteotoxic diseases.
    Keywords:  Charcot Marie Tooth; cGMP; proteasome; protein degradation; proteostasis
    DOI:  https://doi.org/10.1093/brain/awab249
  16. J Biol Chem. 2021 Aug 05. pii: S0021-9258(21)00854-1. [Epub ahead of print] 101052
    A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1.
    Andrew J Boughton, Leonard Liu, Tali Lavy, Oded Kleifeld, David Fushman.
      The ubiquitin-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric ubiquitin chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain ubiquitin-like domains. This degradation pathway is riddled with apparent redundancy - in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photo-crosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and ubiquitin-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the ubiquitin-proteasome system.
    Keywords:  Dsk2; K11-linked polyubiquitin; K48-linked polyubiquitin; Rad23; Rpn1; UBL domain; Ubp6; proteasome; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2021.101052
  17. Mol Cell. 2021 Aug 02. pii: S1097-2765(21)00592-X. [Epub ahead of print]
    The structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client maturation state.
    Kanghyun Lee, Aye C Thwin, Cory M Nadel, Eric Tse, Stephanie N Gates, Jason E Gestwicki, Daniel R Southworth.
      The Hsp90 chaperone promotes folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during folding of kinases, nuclear receptors, and tau. Here we determined the cryoelectron microscopy (cryo-EM) structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å, which, together with mutagenesis and crosslinking analyses, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent to Hsp90 client binding sites, whereas a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 is positioned to act on specific client residues presented during Hsp90-catalyzed remodeling.
    Keywords:  FKBP51; Hsp90; cryo-electron microscopy (cryo-EM); heat shock proteins; immunophilins; molecular chaperones; p23; peptidyl-prolyl isomerase (PPIase)
    DOI:  https://doi.org/10.1016/j.molcel.2021.07.023
  18. FASEB J. 2021 Sep;35(9): e21827
    Atypic SUMOylation of Nor1/NR4A3 regulates neural cell viability and redox sensitivity.
    Jonathan Gagnon, Véronique Caron, Laurent Gyenizse, André Tremblay.
      Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.
    Keywords:  NR4A3; Nor1; SUMO-2; neuroprotection; nuclear receptor; peroxide
    DOI:  https://doi.org/10.1096/fj.202100395R
  19. Cell Death Differ. 2021 Aug 11.
    Dual roles for LUBAC signaling in thymic epithelial cell development and survival.
    Reema Jain, Kelin Zhao, Julie M Sheridan, Melanie Heinlein, Fiona Kupresanin, Waruni Abeysekera, Cathrine Hall, James Rickard, Philippe Bouillet, Henning Walczak, Andreas Strasser, John Silke, Daniel H D Gray.
      Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.
    DOI:  https://doi.org/10.1038/s41418-021-00850-8
  20. Proc Natl Acad Sci U S A. 2021 Aug 17. pii: e2021175118. [Epub ahead of print]118(33):
    Hexokinases inhibit death receptor-dependent apoptosis on the mitochondria.
    Joachim Lauterwasser, Franziska Fimm-Todt, Aline Oelgeklaus, Annabell Schreiner, Kathrin Funk, Hugo Falquez-Medina, Ramona Klesse, Günther Jahreis, Ralf M Zerbes, Katelyn O'Neill, Martin van der Laan, Xu Luo, Frank Edlich.
      Death receptor-mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only protein BID can activate the proapoptotic BCL-2 proteins BAX and BAK and trigger the permeabilization of the mitochondria. BAX and BAK are inhibited by prosurvival BCL-2 proteins through retrotranslocation from the mitochondria into the cytosol, but a specific resistance mechanism to truncated BID-dependent apoptosis is unknown. Here, we report that hexokinase 1 and hexokinase 2 inhibit the apoptosis activator truncated BID as well as the effectors BAX and BAK by retrotranslocation from the mitochondria into the cytosol. BCL-2 protein shuttling and protection from TRAIL- and FasL-induced cell death requires mitochondrial hexokinase localization and interactions with the BH3 motifs of BCL-2 proteins but not glucose phosphorylation. Together, our work establishes hexokinase-dependent retrotranslocation of truncated BID as a selective protective mechanism against death receptor-induced apoptosis on the mitochondria.
    Keywords:  BCL-2 proteins; BH3-only proteins; apoptosis
    DOI:  https://doi.org/10.1073/pnas.2021175118
  21. RSC Chem Biol. 2021 Apr 01. 2(2): 450-467
    Chemical methods for protein site-specific ubiquitination.
    Weijun Gui, Gregory A Davidson, Zhihao Zhuang.
      Ubiquitination is an important protein post-translational modification regulating many cellular processes in eukaryotes. Ubiquitination is catalyzed by a three-enzyme cascade resulting in the conjugation of the C-terminal carboxylate of ubiquitin (Ub) to the ε-amino group of a lysine residue in the acceptor protein via an isopeptide bond. In vitro enzymatic ubiquitination utilizing Ub ligases has been successfully employed to generate Ub dimers and polymers. However, limitations of the enzymatic approach exist, particularly due to the requirement of specific Ub ligase for any given target protein and the low catalytic efficiency of the Ub ligase. To achieve an in-depth understanding of the molecular mechanism of Ub signaling, new methods are needed to generate mono- and poly-ubiquitinated proteins at a specific site with defined polyubiquitin chain linkage and length. Chemical methods offer an attractive solution to the above-described challenges. In this review, we summarize the recently developed chemical methods for generating ubiquitinated proteins using synthetic and semisynthetic approaches. These new tools and approaches, as an important part of the Ub toolbox, are crucial to our understanding and exploitation of the Ub system for novel therapeutics.
    DOI:  https://doi.org/10.1039/d0cb00215a
  22. Sci Rep. 2021 Aug 11. 11(1): 16356
    ATF6 is required for efficient rhodopsin clearance and retinal homeostasis in the P23H rho retinitis pigmentosa mouse model.
    Eun-Jin Lee, Priscilla Chan, Leon Chea, Kyle Kim, Randal J Kaufman, Jonathan H Lin.
      Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho+/P23H) mice. Significantly increased rhodopsin protein levels were found in Atf6-/-Rho+/P23H retinas compared to Atf6+/-Rho+/P23H retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6-/-Rho+/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho+/P23H mice lacking Atf6. By contrast, older Atf6-/-Rho+/P23H mice developed significantly increased retinal degeneration in comparison to Atf6+/-Rho+/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.
    DOI:  https://doi.org/10.1038/s41598-021-95895-7
  23. Mol Cancer Res. 2021 Aug 11. pii: molcanres.0348.2021. [Epub ahead of print]
    N-glycosylation patterns correlate with hepatocellular carcinoma genetic subtypes.
    Andrew DelaCourt, Alyson Black, Peggi Angel, Richard Drake, Yujin Hoshida, Amit Singal, David Lewin, Bachir Taouli, Sara Lewis, Myron Schwartz, M Isabel Fiel, Anand S Mehta.
      Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths globally, and the incidence rate in the US is increasing. Studies have identified inter- and intra-tumor heterogeneity as histological and/or molecular subtypes/variants associated with response to certain molecular targeted therapies. Spatial HCC tissue profiling of N-linked glycosylation by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) may serve as a new method to evaluate the tumor heterogeneity. Previous work has identified significant changes in the N-linked glycosylation of HCC tumors but has not accounted for the heterogeneous genetic and molecular nature of HCC. To determine the correlation between HCC-specific N-glycosylation changes and genetic/molecular tumor features, we profiled HCC tissue samples with MALDI-IMS and correlated the spatial N-glycosylation with a widely used HCC molecular classification (Hoshida subtypes). MALDI-IMS data displayed trends that could approximately distinguish between subtypes, with subtype 1 demonstrating significantly dysregulated N-glycosylation versus adjacent non-tumor tissue. While there were no individual N-glycan structures that could identify specific subtypes, trends emerged regarding the correlation of branched glycan expression to HCC as a whole and fucosylated glycan expression to subtype 1 tumors specifically. Implications: Correlating N-glycosylation to specific subtypes offers the specific detection of subtypes of HCC, which could both enhance early HCC sensitivity and guide targeted clinical therapies.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0348
  24. Cell Chem Biol. 2021 Aug 02. pii: S2451-9456(21)00353-6. [Epub ahead of print]
    Engineered protein-small molecule conjugates empower selective enzyme inhibition.
    Andrew K Lewis, Abbigael Harthorn, Sadie M Johnson, Roy R Lobb, Benjamin J Hackel.
      Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.
    Keywords:  hybrid; ligand; pharmacophore; protein engineering; protein scaffold; yeast display
    DOI:  https://doi.org/10.1016/j.chembiol.2021.07.013
  25. Sci Adv. 2021 Aug;pii: eabi6582. [Epub ahead of print]7(33):
    Structural and biochemical advances on the recruitment of the autophagy-initiating ULK and TBK1 complexes by autophagy receptor NDP52.
    Tao Fu, Mingfang Zhang, Zixuan Zhou, Ping Wu, Chao Peng, Yingli Wang, Xinyu Gong, Ying Li, Yaru Wang, Xiaolong Xu, Miao Li, Liqiang Shen, Lifeng Pan.
      The recruitment of Unc-51-like kinase and TANK-binding kinase 1 complexes is essential for Nuclear dot protein 52-mediated selective autophagy and relies on the specific association of NDP52, RB1-inducible coiled-coil protein 1, and Nak-associated protein 1 (5-azacytidine-induced protein 2, AZI2). However, the underlying molecular mechanism remains elusive. Here, we find that except for the NDP52 SKIP carboxyl homology (SKICH)/RB1CC1 coiled-coil interaction, the LC3-interacting region of NDP52 can directly interact with the RB1CC1 Claw domain, as that of NAP1 FIP200-binding region (FIR). The determined crystal structures of NDP52 SKICH/RB1CC1 complex, NAP1 FIR/RB1CC1 complex, and the related NAP1 FIR/Gamma-aminobutyric acid receptor-associated protein complex not only elucidate the molecular bases underpinning the interactions of RB1CC1 with NDP52 and NAP1 but also reveal that RB1CC1 Claw and Autophagy-related protein 8 family proteins are competitive in binding to NAP1 and NDP52. Overall, our findings provide mechanistic insights into the interactions of NDP52, NAP1 with RB1CC1 and ATG8 family proteins.
    DOI:  https://doi.org/10.1126/sciadv.abi6582
  26. Elife. 2021 Aug 09. pii: e69601. [Epub ahead of print]10
    Hsp40s play complementary roles in the prevention of tau amyloid formation.
    Rose Irwin, Ofrah Faust, Ivana Petrovic, Sharon Grayer Wolf, Hagen Hofmann, Rina Rosenzweig.
      The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles associated with neurodegenerative conditions, such as Alzheimer's disease. In the cell, however, tau aggregation can be prevented by a class of proteins known as molecular chaperones. While numerous chaperones are known to interact with tau, though, little is known regarding the mechanisms by which these prevent tau aggregation. Here, we describe the effects of ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid-fiber formation, and compare these to the small heat-shock protein HSPB1. We find that the chaperones play complementary roles, with each preventing tau aggregation differently and interacting with distinct sets of tau species. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone conformers and even mature fibers. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, distinct site in CTDI. These results lay out the mechanisms by which the diverse members of the Hsp40 family counteract the formation and propagation of toxic tau aggregates, and highlight the fact that chaperones from different families/classes play distinct, yet complementary roles in preventing pathological protein aggregation.
    Keywords:  molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.69601
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