bims-proteo Biomed News
on Proteostasis
Issue of 2023‒03‒26
thirty-six papers selected by
Eric Chevet

  1. bioRxiv. 2023 Mar 09. pii: 2023.03.08.531792. [Epub ahead of print]
      Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.Significance Statement: UFM1 is a ubiquitin-like protein that is selectively conjugated to the large (60S) subunit of ribosomes bound to the endoplasmic reticulum (ER), but the specific biological function of this modification is unclear. Here, we show that UFMylation facilitates proteasome-mediated degradation of arrest polypeptides (APs) which are generated following splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER. We propose that UFMylation weakens the tightly sealed ribosome-translocon junction, thereby allowing the cytosolic ubiquitin-proteasome and ribosome-associated quality control machineries to access ER-APs.
  2. bioRxiv. 2023 Mar 12. pii: 2023.03.11.532186. [Epub ahead of print]
      The integrated stress response (ISR) is a network of eIF2 α kinases, comprising PERK, GCN2, HRI, and PKR, that induce translational and transcriptional signaling in response to diverse insults. The PERK ISR kinase regulates mitochondria in response to endoplasmic reticulum (ER) stress. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activators of other ISR kinases to rescue ISR signaling and promote mitochondrial adaptation in cells lacking PERK. We show that the HRI activator BtdCPU and the GCN2 activator halofuginone activate ISR signaling and restore ER stress sensitivity in Perk- deficient cells. However, these compounds differentially impact mitochondria. BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and ISR activation through the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and altered mitochondrial respiration, mimicking the regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK activity and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly-selective ISR activators.
  3. ACS Chem Biol. 2023 Mar 20.
      Targeted protein degradation (TPD) with proteolysis targeting chimeras (PROTACs), heterobifunctional compounds consisting of protein targeting ligands linked to recruiters of E3 ubiquitin ligases, has arisen as a powerful therapeutic modality to induce the proximity of target proteins with E3 ligases to ubiquitinate and degrade specific proteins in cells. Thus far, PROTACs have primarily exploited the recruitment of E3 ubiquitin ligases or their substrate adapter proteins but have not exploited the recruitment of more core components of the ubiquitin-proteasome system (UPS). In this study, we used covalent chemoproteomic approaches to discover a covalent recruiter against the E2 ubiquitin conjugating enzyme UBE2D─EN67─that targets an allosteric cysteine, C111, without affecting the enzymatic activity of the protein. We demonstrated that this UBE2D recruiter could be used in heterobifunctional degraders to degrade neo-substrate targets in a UBE2D-dependent manner, including BRD4 and the androgen receptor. Overall, our data highlight the potential for the recruitment of core components of the UPS machinery, such as E2 ubiquitin conjugating enzymes, for TPD, and underscore the utility of covalent chemoproteomic strategies for identifying novel recruiters for additional components of the UPS.
  4. Cell Rep. 2023 Mar 10. pii: S2211-1247(23)00297-8. [Epub ahead of print]42(4): 112286
      ER-phagy is a form of autophagy that is mediated by ER-phagy receptors and selectively degrades endoplasmic reticulum (ER). Coronaviruses have been shown to use the ER as a membrane source to establish their double-membrane vesicles (DMVs). However, whether viruses modulate ER-phagy to drive viral DMV formation and its underlying molecular mechanisms remains largely unknown. Here, we demonstrate that coronavirus subverts ER-phagy by hijacking the ER-phagy receptors FAM134B and ATL3 into p62 condensates, resulting in increased viral replication. Mechanistically, we show that viral protein ORF8 binds to and undergoes condensation with p62. FAM134B and ATL3 interact with homodimer of ORF8 and are aggregated into ORF8/p62 liquid droplets, leading to ER-phagy inhibition. ORF8/p62 condensates disrupt ER-phagy to facilitate viral DMV formation and activate ER stress. Together, our data highlight how coronavirus modulates ER-phagy to drive viral replication by hijacking ER-phagy receptors.
    Keywords:  CP: Microbiology; ER stress; ER-phagy; ORF8; SARS-CoV-2; double membrane vesicles; p62 condendation
  5. Mol Cancer Res. 2023 Mar 24. pii: MCR-22-0843. [Epub ahead of print]
      Protein homeostasis (proteostasis) regulates tumor growth and proliferation when cells are exposed to proteotoxic stress, such as during treatment with certain chemotherapeutics. Consequently, cancer cells depend to a greater extent on stress signaling, and require the integrated stress response (ISR), amino acid metabolism, and efficient protein folding and degradation pathways to survive. To define how these interconnected pathways are wired when cancer cells are challenged with proteotoxic stress, we investigated how amino acid abundance influences cell survival when Hsp70, a master proteostasis regulator, is inhibited. We previously demonstrated that cancer cells exposed to a specific Hsp70 inhibitor induce the ISR via the action of two sensors, GCN2 and PERK, in stress-resistant and sensitive cells, respectively. In resistant cells, the induction of GCN2 and autophagy supported resistant cell survival, yet the mechanism by which these events were induced remained unclear. We now report that amino acid availability reconfigures the proteostasis network. Amino acid supplementation, and in particular arginine addition, triggered cancer cell death by blocking autophagy. Consistent with the importance of amino acid availability, which when limited activates GCN2, resistant cancer cells succumbed when challenged with a potentiator for another amino acid sensor, mTORC1, in conjunction with Hsp70 inhibition. Implications: These data position amino acid abundance, GCN2, mTORC1, and autophagy as integrated therapeutic targets whose coordinated modulation regulates the survival of proteotoxic-resistant breast cancer cells.
  6. Semin Cell Dev Biol. 2023 Mar 22. pii: S1084-9521(23)00072-1. [Epub ahead of print]
      Cells entrust ribosomes with the critical task of identifying problematic mRNAs and facilitating their degradation. Ribosomes must communicate when they encounter and stall on an aberrant mRNA, lest they expose the cell to toxic and disease-causing proteins, or they jeopardize ribosome homeostasis and cellular translation. In recent years, ribosomal ubiquitination has emerged as a central signaling step in this process, and proteomic studies across labs and experimental systems show a myriad of ubiquitination sites throughout the ribosome. Work from many labs zeroed in on ubiquitination in one region of the small ribosomal subunit as being functionally significant, with the balance and exact ubiquitination sites determined by stall type, E3 ubiquitin ligases, and deubiquitinases. This review discusses the current literature surrounding ribosomal ubiquitination during translational stress and considers its role in committing translational complexes to decay.
    Keywords:  No-Go mRNA Decay (NGD); Nonstop mRNA Decay (NSD); Ribosome; Translation; Ubiquitin
  7. STAR Protoc. 2023 Mar 24. pii: S2666-1667(23)00157-0. [Epub ahead of print]4(2): 102199
      E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).1.
    Keywords:  Cell Biology; Cell Culture; Cell-based Assays; Molecular Biology; Protein Biochemistry
  8. Antioxid Redox Signal. 2023 Mar 21.
      εβOrganisms adapt to changing environments by engaging cellular stress response pathways that serve to restore proteostasis and enhance survival. A primary adaptive mechanism is the Integrated stress response (ISR), which features phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). Four eIF2α kinases respond to different stresses, enabling cells to rapidly control translation to optimize management of resources and reprogram gene expression for stress adaptation. Phosphorylation of eIF2α blocks its guanine nucleotide exchange factor, eIF2B, thus lowering the levels of eIF2•GTP required to deliver initiator tRNA to ribosomes. While bulk mRNA translation can be sharply lowered by heightened phosphorylation of eIF2α, there are other gene transcripts whose translation is unchanged or preferentially translated. Among the preferentially translated genes is ATF4, which directs transcription of adaptive genes in the ISR. This review focuses on how eIF2α kinases function as first responders of stress, the mechanisms by which eIF2α phosphorylation and other stress signals regulate the exchange activity of eIF2B, and the processes by which the ISR triggers differential mRNA translation. The ISR is tunable and integrates with other stress pathways to optimize adaptation. To illustrate the synergy between stress pathways, we describe the mechanisms and functional significance of communication between the ISR and another key regulator of translation, mTORC1, during acute and chronic amino acid insufficiency. Finally, we discuss the pathological conditions that stem from aberrant regulation of the ISR, as well as therapeutic strategies targeting the ISR to alleviate disease.
  9. Life Sci Alliance. 2023 Jun;pii: e202201419. [Epub ahead of print]6(6):
      Autosomal recessive mutations in the Parkin gene cause Parkinson's disease. Parkin encodes an ubiquitin E3 ligase that functions together with the kinase PINK1 in a mitochondrial quality control pathway. Parkin exists in an inactive conformation mediated by autoinhibitory domain interfaces. Thus, Parkin has become a target for the development of therapeutics that activate its ligase activity. Yet, the extent to which different regions of Parkin can be targeted for activation remained unknown. Here, we have used a rational structure-based approach to design new activating mutations in both human and rat Parkin across interdomain interfaces. Out of 31 mutations tested, we identified 11 activating mutations that all cluster near the RING0:RING2 or REP:RING1 interfaces. The activity of these mutants correlates with reduced thermal stability. Furthermore, three mutations V393D, A401D, and W403A rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extend previous analysis of Parkin activation mutants and suggests that small molecules that would mimic RING0:RING2 or REP:RING1 destabilisation offer therapeutic potential for Parkinson's disease patients harbouring select Parkin mutations.
  10. Am J Physiol Cell Physiol. 2023 Mar 20.
      Ubiquitin regulates a wide variety of biological functions by modifying diverse substrates, via many different conjugation types. Classically, the C-terminus of ubiquitin conjugates to protein substrates via an isopeptide or peptide bond. Recent studies revealed that ubiquitin can form an atypical oxyester-bond, which can target protein and even non-proteinaceous substrates, including sugars and lipids. How non-protein ubiquitination affects substrate and cellular functions is incompletely understood. This review covers recent discoveries in ubiquitination and its potential impacts on biology.
    Keywords:  Ubiquitination; non-protein substrate; oxyester bond; post translational modification; ubiquitin ligase
  11. J Lipid Res. 2023 Mar 21. pii: S0022-2275(23)00035-4. [Epub ahead of print] 100362
      Cholesterol biosynthesis is a highly regulated pathway, with over 20 enzymes controlled at the transcriptional and post-translational level. Whilst some enzymes remain stable, increased sterol levels can trigger degradation of several synthesis enzymes via the ubiquitin-proteasome system. Of note, we previously identified four cholesterol synthesis enzymes as substrates for one E3 ubiquitin ligase, membrane-associated RING-CH-type finger 6 (MARCHF6). Whether MARCHF6 targets the cholesterol synthesis pathway at other points is unknown. In addition, the post-translational regulation of many cholesterol synthesis enzymes, including the C4-demethylation complex (sterol-C4-methyl oxidase-like, SC4MOL; NAD(P) dependent steroid dehydrogenase-like, NSDHL; hydroxysteroid 17-beta dehydrogenase, HSD17B7) is largely uncharacterized. Using cultured mammalian cell-lines (human-derived and Chinese Hamster Ovary cells), we show SC4MOL, the first acting enzyme of C4-demethylation, is a MARCHF6 substrate, and is rapidly turned over and sensitive to sterols. Sterol depletion stabilizes SC4MOL protein levels, whilst sterol excess downregulates both transcript and protein levels. Furthermore, we found SC4MOL depletion by siRNA results in a significant decrease in total cell cholesterol. Thus, our work indicates SC4MOL is the most regulated enzyme in the C4-demethylation complex. Our results further implicate MARCHF6 as a crucial post-translational regulator of cholesterol synthesis, with this E3 ubiquitin ligase controlling levels of at least five enzymes of the pathway.
    Keywords:  C4-demethylation; MARCHF6; NSDHL; SC4MOL; cholesterol biosynthesis; cholesterol metabolism; lipids; molecular biology; post-translational regulation; ubiquitin-proteasome system
  12. Cell Stem Cell. 2023 Mar 16. pii: S1934-5909(23)00071-1. [Epub ahead of print]
      Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. To preserve their fitness, HSCs are particularly dependent on maintaining protein homeostasis (proteostasis). However, how HSCs purge misfolded proteins is unknown. Here, we show that in contrast to most cells that primarily utilize the proteasome to degrade misfolded proteins, HSCs preferentially traffic misfolded proteins to aggresomes in a Bag3-dependent manner and depend on aggrephagy, a selective form of autophagy, to maintain proteostasis in vivo. When autophagy is disabled, HSCs compensate by increasing proteasome activity, but proteostasis is ultimately disrupted as protein aggregates accumulate and HSC function is impaired. Bag3-deficiency blunts aggresome formation in HSCs, resulting in protein aggregate accumulation, myeloid-biased differentiation, and diminished self-renewal activity. Furthermore, HSC aging is associated with a severe loss of aggresomes and reduced autophagic flux. Protein degradation pathways are thus specifically configured in young adult HSCs to preserve proteostasis and fitness but become dysregulated during aging.
    Keywords:  Bag3; aggrephagy; aggresome; aging; autophagy; hematopoietic stem cell; proteasome; protein degradation; proteostasis; stem cell
  13. Elife. 2023 Mar 24. pii: e83159. [Epub ahead of print]12
      Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers. In this study, we set out to identify factors controlling TTP stability. Cellular assays showed that TTP is strongly lysine-ubiquitinated, which is required for its turnover. A genetic screen identified the ubiquitin E3 ligase HUWE1 as a strong regulator of TTP proteasomal degradation, which we found to control TTP stability indirectly by regulating its phosphorylation. Pharmacological assessment of multiple kinases revealed that HUWE1-regulated TTP phosphorylation and stability was independent of the previously characterized effects of MAPK-mediated S52/S178 phosphorylation. HUWE1 function was dependent on phosphatase and E3 ligase binding sites identified in the TTP C-terminus. Our findings indicate that while phosphorylation of S52/S178 is critical for TTP stabilization at earlier times after pro-inflammatory stimulation, phosphorylation of the TTP C-terminus controls its stability at later stages.
    Keywords:  cell biology; e3 ligase; human; huwe1; immunology; inflammation; mouse; tristetraprolin; ubiquitin
  14. Nat Commun. 2023 Mar 23. 14(1): 1625
      Amyloid deposition of the microtubule-associated protein tau is associated with neurodegenerative diseases. In frontotemporal dementia with abnormal tau (FTD-tau), missense mutations in tau enhance its aggregation propensity. Here we describe the structural mechanism for how an FTD-tau S320F mutation drives spontaneous aggregation, integrating data from in vitro, in silico and cellular experiments. We find that S320F stabilizes a local hydrophobic cluster which allosterically exposes the 306VQIVYK311 amyloid motif; identify a suppressor mutation that destabilizes S320F-based hydrophobic clustering reversing the phenotype in vitro and in cells; and computationally engineer spontaneously aggregating tau sequences through optimizing nonpolar clusters surrounding the S320 position. We uncover a mechanism for regulating tau aggregation which balances local nonpolar contacts with long-range interactions that sequester amyloid motifs. Understanding this process may permit control of tau aggregation into structural polymorphs to aid the design of reagents targeting disease-specific tau conformations.
  15. Cell Death Dis. 2023 Mar 22. 14(3): 206
      Parkinson's disease (PD) is characterised by selective death of dopaminergic (DA) neurons in the midbrain and motor function impairment. Gastrointestinal issues often precede motor deficits in PD, indicating that the gut-brain axis is involved in the pathogenesis of this disease. The features of PD include both mitochondrial dysfunction and activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER). PINK1 is a mitochondrial kinase involved in the recycling of defective mitochondria, and PINK1 mutations cause early-onset PD. Like PD patients, pink1 mutant Drosophila show degeneration of DA neurons and intestinal dysfunction. These mutant flies also lack vital proteins due to sustained activation of the kinase R-like endoplasmic reticulum kinase (dPerk), a kinase that induces the UPR. Here, we investigated the role of dPerk in intestinal dysfunction. We showed that intestinal expression of dPerk impairs mitochondrial function, induces cell death, and decreases lifespan. We found that suppressing dPerk in the intestine of pink1-mutant flies rescues intestinal cell death and is neuroprotective. We conclude that in a fly model of PD, blocking gut-brain transmission of UPR-mediated toxicity, is neuroprotective.
  16. FEBS J. 2023 Mar 21.
      Translation elongation becomes arrested when various obstacles arise, such as a series of inefficient rare codons or stable RNA secondary structures, thus, causing ribosomal stalling along the mRNA. Certain wasteful and persistent stalling states are resolved by ribosome rescue pathways. For instance, collisions between stalled and subsequent ribosomes are thought to induce ubiquitination of ribosomal S20 protein by the E3 ubiquitin ligase Hel2, which triggers subsequent rescue reactions. Although structural studies have revealed specific contact sites between collided ribosomes, the ribosomal regions crucial for the rescue reaction remain uncharacterized. In this study, we performed a systematic genetic analysis to identify the molecular regions required for ribosome rescue in Saccharomyces cerevisiae. A series of dominant negative mutations capable of abolishing the rescue reaction were isolated in ribosomal proteins S20 and Asc1. Moreover, mutations in both proteins clustered on the surface of ribosomes between the collided ribosome interfaces, aligned in such a way that they seemingly faced each other. Further analysis via application of the split-TRP1 protein assay, revealed that mutation of either protein distinctively affected the functional interaction between Hel2 and Asc1, suggesting the development of differential functionality at the interface between collided ribosomes. Our results provide novel and complementary insights into the detailed molecular mechanisms of ribosomal rescue pathways.
    Keywords:  collided di-ribosome; functional domain; ribosomal proteins; ribosome rescue
  17. J Chem Inf Model. 2023 Mar 21.
      Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of ∼18 μs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein-protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding.
  18. ACS Chem Biol. 2023 Mar 22.
      Soluble epoxide hydrolase (sEH) is a bifunctional enzyme responsible for lipid metabolism and is a promising drug target. Here, we report the first-in-class PROTAC small-molecule degraders of sEH. Our optimized PROTAC selectively targets the degradation of cytosolic but not peroxisomal sEH, resulting in exquisite spatiotemporal control. Remarkably, our sEH PROTAC molecule has higher potency in cellular assays compared to the parent sEH inhibitor as measured by the significantly reduced ER stress. Interestingly, our mechanistic data indicate that our PROTAC directs the degradation of cytosolic sEH via the lysosome, not through the proteasome. The molecules presented here are useful chemical probes to study the biology of sEH with the potential for therapeutic development. Broadly, our results represent a proof of concept for the superior cellular potency of sEH degradation over sEH enzymatic inhibition, as well as subcellular compartment-selective modulation of a protein by PROTACs.
  19. J Am Chem Soc. 2023 Mar 24.
      The design of PROteolysis-TArgeting Chimeras (PROTACs) requires bringing an E3 ligase into proximity with a target protein to modulate the concentration of the latter through its ubiquitination and degradation. Here, we present a method for generating high-accuracy structural models of E3 ligase-PROTAC-target protein ternary complexes. The method is dependent on two computational innovations: adding a "silent" convolution term to an efficient protein-protein docking program to eliminate protein poses that do not have acceptable linker conformations and clustering models of multiple PROTACs that use the same E3 ligase and target the same protein. Results show that the largest consensus clusters always have high predictive accuracy and that the ensemble of models can be used to predict the dissociation rate and cooperativity of the ternary complex that relate to the degrading activity of the PROTAC. The method is demonstrated by applications to known PROTAC structures and a blind test involving PROTACs against BRAF mutant V600E. The results confirm that PROTACs function by stabilizing a favorable interaction between the E3 ligase and the target protein but do not necessarily exploit the most energetically favorable geometry for interaction between the proteins.
  20. Elife. 2023 Mar 24. pii: e75191. [Epub ahead of print]12
      Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.
    Keywords:  biochemistry; cancer biology; chemical biology; human
  21. bioRxiv. 2023 Mar 07. pii: 2023.03.06.531355. [Epub ahead of print]
      J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8, that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.
  22. Life Sci Alliance. 2023 Jun;pii: e202201805. [Epub ahead of print]6(6):
      Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.
  23. J Cell Sci. 2023 Mar 20. pii: jcs.261139. [Epub ahead of print]
      During host cell invasion, Shigella escapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with autophagy to target Shigella to destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study Shigella septin cage entrapment in its near native state. Quantitative cryo-SXT showed that Shigella fragments mitochondria and enabled visualization of X-ray dense structures (∼30 nm resolution) surrounding Shigella entrapped in septin cages. Using Airyscan confocal microscopy, we observed Lysine 63 (K63)-linked ubiquitin chains decorating septin caged entrapped Shigella. Remarkably, septins and K63 chains form separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy of Shigella. Together, these findings demonstrate how septin caged Shigella are targeted to autophagy and provide fundamental insights into autophagy-cytoskeleton interactions.
    Keywords:   Shigella ; Autophagy; Cryo-SXT; Cytoskeleton; Septins; Ubiquitin
  24. J Biol Chem. 2023 Mar 22. pii: S0021-9258(23)00271-5. [Epub ahead of print] 104629
      O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein Fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat (TPR) region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the TPR recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT.
    Keywords:  O-GlcNAc transferase (OGT); O-linked N-acetylglucosamine (O-GlcNAc); Post-translational modification; glycosylation; intrinsically disordered protein; proteomics
  25. bioRxiv. 2023 Mar 11. pii: 2023.03.10.532109. [Epub ahead of print]
      Stress preconditioning occurs when transient, sublethal stress events impact an organism's ability to counter future stresses. Although preconditioning effects are often noted in the literature, very little is known about the underlying mechanisms. To model preconditioning, we exposed a panel of genetically diverse Drosophila melanogaster to a sublethal heat shock and measured how well the flies survived subsequent exposure to endoplasmic reticulum (ER) stress. The impact of preconditioning varied with genetic background, ranging from dying half as fast to four and a half times faster with preconditioning compared to no preconditioning. Subsequent association and transcriptional analyses revealed that histone methylation, transcriptional regulation, and immune status are all candidate preconditioning modifier pathways. Strikingly, almost all subunits (7/8) in the Set1/COMPASS complex were identified as candidate modifiers of preconditioning. Functional analysis of Set1 knockdown flies demonstrated that loss of Set1 led to the transcriptional dysregulation of canonical ER stress genes during preconditioning. Based on these analyses, we propose a model of preconditioning in which Set1 helps to establish an interim transcriptional 'memory' of previous stress events, resulting in a preconditioned response to subsequent stress.Author Summary: Stress preconditioning occurs when a history of previous stresses impacts an organism's response to subsequent stresses. There are many documented cases of stress preconditioning, but the specific genes and pathways involved in the process are not well understood. Here, we take advantage of the natural genetic variation in the Drosophila Genetic Reference Panel to examine the role genetic variants play in modifying preconditioning outcomes. Our goal is to identify genes that contribute to the underlying mechanisms of preconditioning. Specifically, we measured preconditioning outcomes as the change in death rates of Drosophila on constant endoplasmic reticulum (ER) stress with and without heat stress preconditioning for each strain. We demonstrate that preconditioning outcomes are highly dependent on genetic background. Through association and transcriptional analyses, we found that histone methylation, transcriptional regulation, and immune status are all candidate pathways impacting preconditioning. Functional studies utilizing Set1 knockdown flies demonstrated that Set1, a histone H3 lysine 4 (H3K4) methyltransferase enzyme, is critical for the proper expression of a subset of ER stress genes during preconditioning. Our data indicate that Set1 likely aids in creating a transient transcriptional 'memory' following initial stress that impacts the response to subsequent stress.
  26. Life Sci Alliance. 2023 Jun;pii: e202201710. [Epub ahead of print]6(6):
      The phylum Apicomplexa contains several parasitic species of medical and agricultural importance. The ubiquitination machinery remains, for the most part, uncharacterised in apicomplexan parasites, despite the important roles that it plays in eukaryotic biology. Bioinformatic analysis of the ubiquitination machinery in apicomplexan parasites revealed an expanded ovarian tumour domain-containing (OTU) deubiquitinase (DUB) family in Toxoplasma, potentially reflecting functional importance in apicomplexan parasites. This study presents comprehensive characterisation of Toxoplasma OTU DUBs. AlphaFold-guided structural analysis not only confirmed functional orthologues found across eukaryotes, but also identified apicomplexan-specific enzymes, subsequently enabling discovery of a cryptic OTU DUB in Plasmodium species. Comprehensive biochemical characterisation of 11 Toxoplasma OTU DUBs revealed activity against ubiquitin- and NEDD8-based substrates and revealed ubiquitin linkage preferences for Lys6-, Lys11-, Lys48-, and Lys63-linked chain types. We show that accessory domains in Toxoplasma OTU DUBs impose linkage preferences, and in case of apicomplexan-specific TgOTU9, we discover a cryptic ubiquitin-binding domain that is essential for TgOTU9 activity. Using the auxin-inducible degron (AID) to generate knockdown parasite lines, TgOTUD6B was found to be important for Toxoplasma growth.
  27. Sci Adv. 2023 Mar 22. 9(12): eade1851
      Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3β, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.
  28. Cell Rep. 2023 Mar 20. pii: S2211-1247(23)00283-8. [Epub ahead of print]42(3): 112272
      RNA virus infection usually triggers a range of host immune responses, including the induction of proinflammatory cytokines, interferons, and interferon-stimulated genes (ISGs). Here, we report that UBL7, a ubiquitin-like protein, is upregulated during RNA virus infection and induced by type I interferon as an ISG. UBL7-deficient mice exhibit increased susceptibility to viral infection due to attenuated antiviral innate immunity. UBL7 enhances innate immune response to viral infection by promoting the K27-linked polyubiquitination of MAVS. UBL7 interacts with TRIM21, an E3 ubiquitin ligase of MAVS, and promotes the combination of TRIM21 with MAVS in a dose-dependent manner, facilitating the K27-linked polyubiquitination of MAVS and recruiting of TBK1 to enhance the IFN signaling pathway. Moreover, UBL7 has a broad-spectrum antiviral function as an immunomodulatory adaptor protein. Therefore, UBL7 positively regulates innate antiviral signaling and promotes positive feedback to enhance and amplify the antiviral response.
    Keywords:  CP: Immunology; Innate immunity; Interferon-stimulated genes; K27-linked polyubiquitination; MAVS; TRIM21; UBL7
  29. J Cell Biol. 2023 Jun 05. pii: e202112101. [Epub ahead of print]222(6):
      Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP.
  30. Mol Cell. 2023 Mar 08. pii: S1097-2765(23)00151-X. [Epub ahead of print]
      SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.
    Keywords:  ALKBH5; MYC; SF3B1; acute myeloid leukemia; alternative splicing; genome integrity; m(6)A; myelodysplastic syndromes; p53; translation
  31. Immunity. 2023 Mar 14. pii: S1074-7613(23)00088-2. [Epub ahead of print]
      NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.
    Keywords:  ATPase; NLRC4; NLRP3; NLRs; PELO; catalyzer; inflammasome; oligomerization
  32. bioRxiv. 2023 Mar 09. pii: 2023.03.09.531795. [Epub ahead of print]
      Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations are redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. Depending on the type of mitochondrial mutation, certain mechanisms can efficiently rescue cell death vulnerability. One method is the inhibition of mitochondrial translation elongation using tetracyclines, potent suppressors of cell death in mitochondrial disease mutant cells. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that in mitochondrial mutant disease cells, tetracycline-mediated inhibition of mitoribosome elongation promotes survival through suppression of the ER stress IRE1α protein. Tetracyclines increased levels of the splitting factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) at the mitochondria with recruitment to the mitochondrial ribosome (mitoribosome) large subunit. MALSU1, but not other quality control factors, was required for tetracycline-induced cell survival in mitochondrial disease mutant cells during glucose starvation. In these cells, nutrient stress induced cell death through IRE1α activation associated with a strong protein loading in the ER lumen. Notably, tetracyclines rescued cell death through suppression of IRE1α oligomerization and activity. Consistent with MALSU1 requirement, MALSU1 deficient mitochondrial mutant cells were sensitive to glucose-deprivation and exhibited increased ER stress and activation of IRE1α that was not reversed by tetracyclines. These studies show that inhibition of mitoribosome elongation signals to the ER to promote survival, establishing a new interorganelle communication between the mitoribosome and ER with implications in basic mechanisms of cell survival and treatment of mitochondrial diseases.Significance Statement: Mitochondrial diseases are a rare and heterogenous class of diseases that result from mutations in mitochondrial genes. Currently, there are no curative therapies due to a lack of mechanistic insights into pathological transformation and signaling. Our lab has discovered that the class of mitochondrial ribosome targeting antibiotics, tetracyclines, promote survival and fitness in models of mitochondrial disease, establishing a new paradigm of cell survival under nutrient stress conditions. In the current study, we present mechanistic insights into tetracyclines ability to rescue mitochondrial disease cells, detailing an interorganelle communication between mitochondrial protein translation and the unfolded protein response during endoplasmic reticulum stress.
  33. Nucleic Acids Res. 2023 Mar 23. pii: gkad194. [Epub ahead of print]
      The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and disease. However, the inability to accurately visualise these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualise post-transcriptional modifications within the 18S rRNA and four post-translational modifications of ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilisation and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unravelling the functional role of ribosomal RNA modifications.
  34. Biochim Biophys Acta Gene Regul Mech. 2023 Mar 16. pii: S1874-9399(23)00022-6. [Epub ahead of print]1866(2): 194927
      N6-methyladenosine (m6A) plays an important role in regulating many physiological and disease processes in vertebrates, in which methyltransferase-like 3 (METTL3) is the best-known m6A methyltransferase. However, the functional roles of invertebrate METTL3 have not yet been highlighted. In this study, we found that METTL3 from Apostichopus japonicus (AjMETTL3) was significantly induced in coelomocytes accompanied by higher levels of m6A modification in response to Vibrio splendidus challenge. Overexpression or silencing of AjMETTL3 in coelomocytes increased or decreased the m6A levels and promoted or inhibited V. splendidus-induced coelomocyte apoptosis, respectively. To further explore the molecular mechanism of AjMETTL3-mediated coelomic immunity, m6A-seq analysis revealed that the endoplasmic reticulum-related degradation (ERAD) pathway was significantly enriched, in which suppressor/enhancer of Lin-12-like (AjSEL1L) was suggested to be a target of AjMETTL3 in a negative regulatory manner. Functional analysis revealed that the increased AjMETTL3 reduced the stability of AjSEL1L mRNA by targeting the m6A modification site of 2004 bp-GGACA-2008 bp. The decreased AjSEL1L was further confirmed to be involved in AjMETTL3-mediated coelomocyte apoptosis. Mechanistically, the inhibited AjSEL1L increased the transcription of AjOS9 and Ajp97 in the EARD pathway to promote ubiquitin protein accumulation and ER stress, which further activated AjPERK-AjeIF2α pathway dependent coelomocyte apoptosis, but not the AjIRE1 or AjATF6 pathway. Taken together, our results supported invertebrate METTL3-mediated coelomocyte apoptosis by regulating the PERK-eIF2α pathway.
    Keywords:  Apoptosis; Methyltransferase-like 3; N6-methyladenosine; Protein kinase RNA-like endoplasmic reticulum kinase; Suppressor/enhancer of Lin-12-like
  35. Cancer Res Commun. 2023 Mar;3(3): 459-470
      miRNA biogenesis is a cellular process that produces mature miRNAs from their primary transcripts, pri-miRNAs, via two RNAse III enzyme complexes: the Drosha-DGCR8 microprocessor complex in the nucleus and the Dicer-TRBP complex in the cytoplasm. Emerging evidence suggests that miRNA biogenesis is tightly regulated by posttranscriptional and posttranslational modifications and aberrant miRNA biogenesis is associated with various human diseases including cancer. DGCR8 has been shown to be modified by SUMOylation. Yet, the SUMO ligase mediating DGCR8 SUMOylation is currently unknown. Here, we report that USP36, a nucleolar ubiquitin-specific protease essential for ribosome biogenesis, is a novel regulator of DGCR8. USP36 interacts with the microprocessor complex and promotes DGCR8 SUMOylation, specifically modified by SUMO2. USP36-mediated SUMOylation does not affect the levels of DGCR8 and the formation of the Drosha-DGCR8 complex, but promotes the binding of DGCR8 to pri-miRNAs. Consistently, abolishing DGCR8 SUMOylation significantly attenuates its binding to pri-miRNAs and knockdown of USP36 attenuates pri-miRNA processing, resulting in marked reduction of tested mature miRNAs. Induced expression of a SUMOylation-defective mutant of DGCR8 inhibits cell proliferation. Together, these results suggest that USP36 plays an important role in regulating miRNA biogenesis by SUMOylating DGCR8.Significance: This study identifies that USP36 mediates DGCR8 SUMOylation by SUMO2 and is critical for miRNA biogenesis. As USP36 is frequently overexpressed in various human cancers, our study suggests that deregulated USP36-miRNA biogenesis pathway may contribute to tumorigenesis.
  36. Nat Microbiol. 2023 Mar 23.
      Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR-Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.