bims-proteo Biomed News
on Proteostasis
Issue of 2021‒03‒14
38 papers selected by
Eric Chevet

  1. EMBO Rep. 2021 Mar 12. e51412
      In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).
    Keywords:  ER stress; ERAD; cancer; endoplasmic reticulum; reflux
  2. Genetics. 2021 Mar 03. 217(1): 1-19
      Intracellular proteolysis by the ubiquitin-proteasome system regulates numerous processes and contributes to protein quality control (PQC) in all eukaryotes. Covalent attachment of ubiquitin to other proteins is specified by the many ubiquitin ligases (E3s) expressed in cells. Here we determine the E3s in Saccharomyces cerevisiae that function in degradation of proteins bearing various PQC degradation signals (degrons). The E3 Ubr1 can function redundantly with several E3s, including nuclear-localized San1, endoplasmic reticulum/nuclear membrane-embedded Doa10, and chromatin-associated Slx5/Slx8. Notably, multiple degrons are targeted by more ubiquitylation pathways if directed to the nucleus. Degrons initially assigned as exclusive substrates of Doa10 were targeted by Doa10, San1, and Ubr1 when directed to the nucleus. By contrast, very short hydrophobic degrons-typical targets of San1-are shown here to be targeted by Ubr1 and/or San1, but not Doa10. Thus, distinct types of PQC substrates are differentially recognized by the ubiquitin system in a compartment-specific manner. In human cells, a representative short hydrophobic degron appended to the C-terminus of GFP-reduced protein levels compared with GFP alone, consistent with a recent study that found numerous natural hydrophobic C-termini of human proteins can act as degrons. We also report results of bioinformatic analyses of potential human C-terminal degrons, which reveal that most peptide substrates of Cullin-RING ligases (CRLs) are of low hydrophobicity, consistent with previous data showing CRLs target degrons with specific sequences. These studies expand our understanding of PQC in yeast and human cells, including the distinct but overlapping PQC E3 substrate specificity of the cytoplasm and nucleus.
    Keywords:  degron; proteasome; protein degradation; protein quality control; ubiquitin
  3. Biochim Biophys Acta Mol Cell Res. 2021 Mar 08. pii: S0167-4889(21)00055-0. [Epub ahead of print] 119001
      Endoplasmic Reticulum (ER) stress signaling is an adaptive mechanism triggered when protein folding demand overcomes the folding capacity of this compartment, thereby leading to the accumulation of improperly folded proteins. This stress signaling pathway is named the Unfolded Protein Response (UPR) and aims at restoring ER homeostasis. However, if this fails, mechanisms orienting cells towards death processes are initiated. Herein, we summarize the most recent findings connecting ER stress and the UPR with identified death mechanisms including apoptosis, necrosis, pyroptosis, ferroptosis, and autophagy. We highlight new avenues that could be investigated and controlled through actionable mechanisms in physiology and pathology.
    Keywords:  Apoptosis; Autophagy; Cell death; Endoplasmic reticulum; Ferroptosis; Pyroptosis; Unfolded protein response
  4. Front Plant Sci. 2021 ;12 639625
      Jasmonates (JA) are oxylipin-derived phytohormones that trigger the production of specialized metabolites that often serve in defense against biotic stresses. In Medicago truncatula, a JA-induced endoplasmic reticulum-associated degradation (ERAD)-type machinery manages the production of bioactive triterpenes and thereby secures correct plant metabolism, growth, and development. This machinery involves the conserved RING membrane-anchor (RMA)-type E3 ubiquitin ligase MAKIBISHI1 (MKB1). Here, we discovered two additional members of this protein control apparatus via a yeast-based protein-protein interaction screen and characterized their function. First, a cognate E2 ubiquitin-conjugating enzyme was identified that interacts with MKB1 to deliver activated ubiquitin and to mediate its ubiquitination activity. Second, we identified a heat shock protein 40 (HSP40) that interacts with MKB1 to support its activity and was therefore designated MKB1-supporting HSP40 (MASH). MASH expression was found to be co-regulated with that of MKB1. The presence of MASH is critical for MKB1 and ERAD functioning because the dramatic morphological, transcriptional, and metabolic phenotype of MKB1 knock-down M. truncatula hairy roots was phenocopied by silencing of MASH. Interaction was also observed between the Arabidopsis thaliana (Arabidopsis) homologs of MASH and MKB1, suggesting that MASH represents an essential and plant-specific component of this vital and conserved eukaryotic protein quality control machinery.
    Keywords:  3-hydroxy-3-methylglutaryl-CoA reductase; E3-ubiquitin ligase; RING membrane-anchor protein; chaperone; endoplasmic reticulum; jasmonate; protein quality control; triterpene saponin
  5. J Proteomics. 2021 Mar 08. pii: S1874-3919(21)00081-6. [Epub ahead of print] 104182
      Protein aggregation is indicative of failing protein quality control systems. These systems are responsible for the refolding or degradation of aberrant and misfolded proteins. Heat stress can cause proteins to misfold, triggering cellular responses and a marked increase in the ubiquitination of proteins. This response has been characterized in yeast, however more studies are needed within mammalian cells. Herein, we examine proteins that become ubiquitinated during heat shock in human tissue culture cells using diGly enrichment coupled with mass spectrometry. A majority of these proteins are localized in the nucleus or cytosol. Proteins which are conjugated under stress display longer sequence lengths, more interaction partners, and more hydrophobic patches than controls but do not show lower melting temperatures. Furthermore, heat-induced conjugation sites occur less frequently in disordered regions and are closer to hydrophobic patches than other ubiquitination sites; perhaps providing novel insight into the molecular mechanism mediating this response. Nuclear and cytosolic pools of modified proteins appear to have different protein features. Using a pulse-SILAC approach, we found that both long-lived and newly-synthesized proteins are conjugated under stress. Modified long-lived proteins are predominately nuclear and were distinct from newly-synthesized proteins, indicating that different pathways may mediate the heat-induced increase of polyubiquitination. SIGNIFICANCE: The maintenance of protein homeostasis requires a balance of protein synthesis, folding, and degradation. Under stress conditions, the cell must rapidly adapt by increasing its folding capacity to eliminate aberrant proteins. A major pathway for proteolysis is mediated by the ubiquitin proteasome system. While increased ubiquitination after heat stress was observed over 30 years ago, it remains unclear which proteins are conjugated during heat shock in mammalian cells and by what means this conjugation occurs. In this study, we combined SILAC-based mass spectrometry with computational analyses to reveal features associated to proteins ubiquitinated while under heat shock. Interestingly, we found that conjugation sites induced by the stress are less often located within disordered regions and more often located near hydrophobic patches. Our study showcases how proteomics can reveal distinct feature associated to a cohort of proteins that are modified post translationally and how the ubiquitin conjugation sites are preferably selected in these conditions. Our work opens a new path for delineating the molecular mechanisms leading to the heat stress response and the regulation of protein homeostasis.
    Keywords:  Heat shock; Intrinsic protein disorder; Mass spectrometry; Proteostasis; SILAC; Ubiquitin
  6. Elife. 2021 Mar 10. pii: e65703. [Epub ahead of print]10
      The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.
    Keywords:  biochemistry; cell biology; chemical biology; human
  7. Trends Cell Biol. 2021 Mar 05. pii: S0962-8924(21)00029-5. [Epub ahead of print]
      The biosynthesis of about one third of the human proteome, including membrane receptors and secreted proteins, occurs in the endoplasmic reticulum (ER). Conditions that perturb ER homeostasis activate the unfolded protein response (UPR). An 'optimistic' UPR output aims at restoring homeostasis by reinforcement of machineries that guarantee efficiency and fidelity of protein biogenesis in the ER. Yet, once the UPR 'deems' that ER homeostatic readjustment fails, it transitions to a 'pessimistic' output, which, depending on the cell type, will result in apoptosis. In this article, we discuss emerging concepts on how the UPR 'evaluates' ER stress, how the UPR is repurposed, in particular in B cells, and how UPR-driven counter-selection of cells undergoing homeostatic failure serves organismal homeostasis and humoral immunity.
    Keywords:  B cell development; RIDD; antibody production; endoplasmic reticulum; proteostasis; unfolded protein response
  8. Cell Rep. 2021 Mar 09. pii: S2211-1247(21)00140-6. [Epub ahead of print]34(10): 108826
      A major pathway for proinflammatory protein release by macrophages is inflammasome-mediated pyroptotic cell death. As conventional secretion, unconventional secretion, and cell death are executed simultaneously, however, the cellular mechanisms regulating this complex paracrine program remain incompletely understood. Here, we devise a quantitative proteomics strategy to define the cellular exit route for each protein by pharmacological and genetic dissection of cellular checkpoints regulating protein release. We report the release of hundreds of proteins during pyroptosis, predominantly due to cell lysis. They comprise constitutively expressed and transcriptionally induced proteins derived from the cytoplasm and specific intracellular organelles. Many low-molecular-weight proteins including the cytokine interleukin-1β, alarmins, and lysosomal-cargo proteins exit cells in the absence of cell lysis. Cytokines and alarmins are released in an endoplasmic reticulum (ER)-Golgi-dependent manner as free proteins rather than by extracellular vesicles. Our work provides an experimental framework for the dissection of cellular exit pathways and a resource for pyroptotic protein release.
    Keywords:  NLRP3; TLR4; cell death; gasdermin D; macrophage; mass spectrometry; protein secretion; proteomics; pyroptosis; secretome
  9. Endocr Rev. 2021 Mar 08. pii: bnab006. [Epub ahead of print]
      The endoplasmic reticulum (ER) hosts linear polypeptides and fosters natural folding of proteins through ER-residing chaperones and enzymes. Failure of the ER to align and compose proper protein architecture leads to accumulation of misfolded/unfolded proteins in the ER lumen, which disturbs ER homeostasis to provoke ER stress. Presence of ER stress initiates the cytoprotective unfolded protein response (UPR) to restore ER homeostasis or instigates a rather maladaptive UPR to promote cell death. Although a wide array of cellular processes such as persistent autophagy, dysregulated mitophagy, and secretion of pro-inflammatory cytokines may contribute to the onset and progression of cardiometabolic diseases, it is well perceived that ER stress also evokes onset and development of cardiometabolic diseases, particularly, cardiovascular diseases, diabetes mellitus, obesity, and chronic kidney disease. Meanwhile, these pathological conditions further aggravate ER stress, creating a rather vicious cycle. Here in this review, we aimed at summarizing and updating the available information on ER stress in cardiovascular diseases, diabetes mellitus, obesity, and chronic kidney disease, hoping to offer novel insights for the management of these cardiometabolic comorbidities through regulation of ER stress.
    Keywords:  ER stress; cardiometabolic disease; chronic kidney disease; diabetes; obesity
  10. Cell Death Differ. 2021 Mar 12.
      In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.
  11. Curr Opin Cell Biol. 2021 Mar 09. pii: S0955-0674(21)00016-8. [Epub ahead of print]71 95-102
      Biomolecules in the secretory pathway use membrane trafficking for reaching their final intracellular destination or for secretion outside the cell. This highly dynamic and multipartite process involves different organelles that communicate to one another while maintaining their identity, shape, and function. Recent studies unraveled new mechanisms of interorganelle communication that help organize the early secretory pathway. We highlight how the spatial proximity between endoplasmic reticulum (ER) exit sites and early Golgi elements provides novel means of ER-Golgi communication for ER export. We also review recent findings on how membrane contact sites between the ER and the trans-Golgi membranes can sustain anterograde traffic out of the Golgi complex.
    Keywords:  ER exit sites; Golgi complex; Membrane contact sites; Membrane trafficking; Organelle morphology
  12. J Cell Sci. 2021 Mar 12. pii: jcs.254201. [Epub ahead of print]
      The recognition and disposal of misfolded proteins is essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. The p97 AAA-ATPase in combination with adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the proteasome or autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes, membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates aggresome formation and clearance and identify a novel role for the adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to aggresomes and UBXN1 knockout cells are unable to form aggresomes. Loss of p97-UBXN1 results in increased Huntingtin polyQ inclusion bodies both in mammalian cells as well as in a C.elegans model of Huntington's Disease. Together our work identifies evolutionarily conserved roles for p97-UBXN1 in the disposal of protein aggregates.
    Keywords:  Aggregate; Aggresome; Inclusion body; PolyQ; Proteasome; Ubiquitin
  13. Trends Cell Biol. 2021 Mar 04. pii: S0962-8924(21)00028-3. [Epub ahead of print]
      Organelles cooperate with each other to control cellular homeostasis and cell functions by forming close connections through membrane contact sites. Important contacts are present between the endoplasmic reticulum (ER), the main intracellular Ca2+-storage organelle, and the mitochondria, the organelle responsible not only for the majority of cellular ATP production but also for switching on cell death processes. Several Ca2+-transport systems focalize at these contact sites, thereby enabling the efficient transmission of Ca2+ signals from the ER toward mitochondria. This provides tight control of mitochondrial functions at the microdomain level. Here, we discuss how ER-mitochondrial Ca2+ transfers support cell function and how their dysregulation underlies, drives, or contributes to pathogenesis and pathophysiology, with a major focus on cancer and neurodegeneration but also with attention to other diseases such as diabetes and rare genetic diseases.
    Keywords:  Ca(2+) signaling; MAMs; cancer; contact sites; genetic diseases; neurodegeneration
  14. Curr Biol. 2021 Mar 05. pii: S0960-9822(21)00275-X. [Epub ahead of print]
      The oxidative environment within the mitochondria makes them particularly vulnerable to proteotoxic stress. To maintain a healthy mitochondrial network, eukaryotes have evolved multi-tiered quality control pathways. If the stress cannot be alleviated, defective mitochondria are selectively removed by autophagy via a process termed mitophagy. Despite significant advances in metazoans and yeast, in plants, the molecular underpinnings of mitophagy are largely unknown. Here, using time-lapse imaging, electron tomography, and biochemical assays, we show that uncoupler treatments cause loss of mitochondrial membrane potential and induce autophagy in Arabidopsis. The damaged mitochondria are selectively engulfed by autophagosomes that are labeled by ATG8 proteins in an ATG5-dependent manner. Friendly, a member of the clustered mitochondria protein family, is recruited to the damaged mitochondria to mediate mitophagy. In addition to the stress, mitophagy is also induced during de-etiolation, a major cellular transformation during photomorphogenesis that involves chloroplast biogenesis. De-etiolation-triggered mitophagy is involved in cotyledon greening, pointing toward an inter-organellar crosstalk mechanism. Altogether, our results demonstrate how plants employ mitophagy to recycle damaged mitochondria during stress and development.
    Keywords:  Arabidopsis; autophagy; clustered mitochondria protein; de-etiolation; electron tomography; mitochondria recycling; mitophagy; time-lapse live-cell imaging; uncoupler
  15. Cell Stem Cell. 2021 Mar 04. pii: S1934-5909(21)00058-8. [Epub ahead of print]
      Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.
    Keywords:  HSC regeneration; HectD1; Polypeptide exit tunnel; ZNF622; hematopoietic stem cells; protein synthesis; ribosome assembly; ribosome biogenesis; signaling; ubiquitin
  16. PLoS Biol. 2021 Mar;19(3): e3001096
      The regulation of protein synthesis is essential for maintaining cellular homeostasis, especially during stress responses, and its dysregulation could underlie the development of human diseases. The critical step during translation regulation is the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α). Here we report the identification of a direct kinase of eIF2α, microtubule affinity-regulating kinase 2 (MARK2), which phosphorylates eIF2α in response to proteotoxic stress. The activity of MARK2 was confirmed in the cells lacking the 4 previously known eIF2α kinases. MARK2 itself was found to be a substrate of protein kinase C delta (PKCδ), which serves as a sensor for protein misfolding stress through a dynamic interaction with heat shock protein 90 (HSP90). Both MARK2 and PKCδ are activated via phosphorylation in proteotoxicity-associated neurodegenerative mouse models and in human patients with amyotrophic lateral sclerosis (ALS). These results reveal a PKCδ-MARK2-eIF2α cascade that may play a critical role in cellular proteotoxic stress responses and human diseases.
  17. Nat Cell Biol. 2021 Mar 08.
      Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.
  18. Cell Rep. 2021 Mar 09. pii: S2211-1247(21)00141-8. [Epub ahead of print]34(10): 108827
      Calcium transfer from the endoplasmic reticulum (ER) to mitochondria is a critical contributor to apoptosis. B cell lymphoma 2 (BCL-2) ovarian killer (BOK) localizes to the ER and binds the inositol 1,4,5-trisphosophate receptor (IP3R). Here, we show that BOK is necessary for baseline mitochondrial calcium levels and stimulus-induced calcium transfer from the ER to the mitochondria. Murine embryonic fibroblasts deficient for BOK have decreased proximity of the ER to the mitochondria and altered protein composition of mitochondria-associated membranes (MAMs), which form essential calcium microdomains. Rescue of the ER-mitochondrial juxtaposition with drug-inducible interorganelle linkers reveals a kinetic disruption, which when overcome in Bok-/- cells is still insufficient to rescue thapsigargin-induced calcium transfer and apoptosis. Likewise, a BOK mutant unable to interact with IP3R restores ER-mitochondrial proximity, but not ER-mitochondrial calcium transfer, MAM protein composition, or apoptosis. This work identifies the dynamic coordination of ER-mitochondrial contact by BOK as an important control point for apoptosis.
    Keywords:  BCL-2 family; BOK; IP3R; MAMs; MERCs; apoptosis; calcium; endoplasmic reticulum; mitochondria-ER contact sites; mitochondria-associated membranes
  19. Cell Stress. 2021 Feb 17. 5(3): 33-36
      The autophagy-lysosomal pathway is one of the main degradative routes which cells use to balance sources of energy. A number of proteins orchestrate the formation of autophagosomes, membranous organelles instrumental in autophagy. Selective autophagy, involving the recognition and removal of specific targets, is mediated by autophagy receptors, which recognize cargos and the autophagosomal membrane protein LC3 for lysosomal degradation. Recently, bidirectional crosstalk has emerged between autophagy and primary cilia, microtubule-based sensory organelles extending from cells and anchored by the basal body, derived from the mother centriole of the centrosome. The molecular mechanisms underlying the direct role of autophagic proteins in cilia biology and, conversely, the impact of this organelle in autophagy remains elusive. Recently, we uncovered the molecular mechanism by which the centrosomal/basal body protein OFD1 controls the LC3-mediated autophagic cascade. In particular, we demonstrated that OFD1 acts as a selective autophagy receptor by regulating the turnover of unc-51-like kinase (ULK1) complex, which plays a crucial role in the initiation steps of autophagosome biogenesis. Moreover, we showed that patients with a genetic condition caused by mutations in OFD1 and associated with cilia dysfunction, display excessive autophagy and we demonstrated that autophagy inhibition significantly ameliorates the renal cystic phenotype in a conditional mouse model recapitulating the features of the disease (Morleo et al. 2020, EMBO J, doi: 10.15252/embj.2020105120). We speculate that abnormal autophagy may underlie some of the clinical manifestations observed in the disorders ascribed to cilia dysfunction.
    Keywords:  Autophagy receptor; OFD1; Oral-Facial-Digital type I syndrome; Primary cilium; Renal cystic disease; Selective autophagy; ULK1 complex
  20. Life Sci Alliance. 2021 May;pii: e202000927. [Epub ahead of print]4(5):
      Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslational modifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immunofluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.
  21. J Biol Chem. 2021 Mar 03. pii: S0021-9258(21)00281-7. [Epub ahead of print] 100505
      Low levels of oxygen (hypoxia) occurs in many (patho)physiological situations. Adaptation to hypoxia is in part mediated by proteins expressed in the extracellular space that mature in the endoplasmic reticulum (ER) prior to traversing the secretory pathway. The majority of such ER cargo require disulfide bonds for structural stability. Disulfide bonds are formed co- and post-translationally in a redox relay that requires a terminal electron acceptor like oxygen. We have previously demonstrated that some ER cargo proteins like low-density lipoprotein receptor (LDLR) and influenza hemagglutinin (Flu-HA) are unable to complete disulfide bond formation in the absence of oxygen, limiting their ability to pass ER quality control and their ultimate expression. Here, using radioactive pulse-chase immunoprecipitation analysis, we demonstrate that hypoxia-induced ER cargo proteins like carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A) complete disulfide bond formation and mature with similar kinetics under hypoxia and normoxia. A global in silico analysis of ER cargo revealed that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other ER cargo proteins. These data demonstrate the existence of alternative electron acceptors to oxygen for disulfide bond formation in cellulo. However, the ability of different proteins to utilize an oxygen-independent pathway for disulfide bond formation varies widely, contributing to differential gene expression in hypoxia. The superior ability of hypoxia-induced proteins like VEGF-A and CA9 to mature in hypoxia may be conferred by a simpler disulfide architecture.
    Keywords:  Carbonic anhydrase 9 (CA9); disulfide; endoplasmic reticulum (ER); glycosylation; hypoxia; low-density lipoprotein receptor (LDLR); protein folding; tumor microenvironment; vascular endothelial growth factor (VEGF)
  22. Glia. 2021 Mar 11.
      Reactive astrogliosis is a pathological feature of spinal cord injury (SCI). The ubiquitin-proteasome system plays a crucial role in maintaining protein homeostasis and has been widely studied in neuroscience. Little, however, is known about the underlying function of deubiquitinating enzymes in reactive astrogliosis following SCI. Here, we found that ubiquitin-specific protease 18 (USP18) was significantly upregulated in astrocytes following scratch injury, and in the injured spinal cord in mice. Knockdown of USP18 in vitro and conditional knockout of USP18 in astrocytes (USP18 CKO) in vivo significantly attenuated reactive astrogliosis. In mice, this led to widespread inflammation and poor functional recovery following SCI. In contrast, overexpression of USP18 in mice injected with adeno-associated virus (AAV)-USP18 had beneficial effects following SCI. We showed that USP18 binds, deubiquitinates, and thus, stabilizes SRY-box transcription factor 9 (SOX9), thereby regulating reactive astrogliosis. We also showed that the Hedgehog (Hh) signaling pathway induces expression of USP18 through Gli2-mediated transcriptional activation after SCI. Administration of the Hh pathway activator SAG significantly increased reactive astrogliosis, reduced lesion area and promoted functional recovery in mice following SCI. Our results demonstrate that USP18 positively regulates reactive astrogliosis by stabilizing SOX9 and identify USP18 as a promising target for the treatment of SCI.
    Keywords:  Gli2; SOX9; USP18; reactive astrogliosis; spinal cord injury
  23. Sci Rep. 2021 Mar 08. 11(1): 5429
      Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.
  24. Methods Mol Biol. 2021 Mar 11.
      The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has proven to be a powerful system creating new opportunities to interrogate molecular mechanisms controlling cell fate determination. Under standard conditions, the generation of iPSCs upon overexpression of OCT4, SOX2, KLF4, and c-MYC (OSKM) is generally slow and inefficient due to the presence of barriers that confer resistance to cell fate changes. Hyperactivated endoplasmic reticulum (ER) stress has emerged as a major reprogramming barrier that impedes the initial mesenchymal-to-epithelial transition (MET) step to form iPSCs from mesenchymal somatic cells. Here, we describe several systems to detect ER stress in the context of OSKM reprogramming and chemical interventions to modulate this process for improving iPSC formation.
    Keywords:  Cell fate change; Endoplasmic reticulum stress; Induced pluripotent stem cell; Mesenchymal-to-epithelial transition; Pluripotency; Unfolded protein response
  25. Biochem Biophys Res Commun. 2021 Mar 08. pii: S0006-291X(21)00363-6. [Epub ahead of print]550 184-190
      Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. Here, we investigated the role of linear ubiquitin E3 ligase (LUBEL), a DrosophilaHOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, we propose a new role of linear ubiquitination in proteostasis in the muscle aging.
    Keywords:  Aging; Drosophila; LUBEL; Linear ubiquitination; Muscle; Proteostasis
  26. Front Immunol. 2021 ;12 646633
      Conjugation with the small ubiquitin-like modifier (SUMO) constitutes a key post-translational modification regulating the stability, activity, and subcellular localization of its target proteins. However, the vast numbers of identified SUMO substrates obscure a clear view on the function of SUMOylation in health and disease. This article presents a comprehensive review on the physiological relevance of SUMOylation by discussing how global SUMOylation levels-rather than specific protein SUMOylation-shapes the immune response. In particular, we highlight the growing body of work on SUMOylation in intestinal pathologies, because of the unique metabolic, infectious, and inflammatory challenges of this organ. Recent studies show that global SUMOylation can help restrain detrimental inflammation while maintaining immune defenses and tissue integrity. These results warrant further efforts to develop new therapeutic tools and strategies to control SUMOylation in infectious and inflammatory disorders.
    Keywords:  adaptive response mechanism; cell stress response; intestinal pathologies; post-translational modification; small ubiquitin like modifier
  27. Database (Oxford). 2021 Mar 08. pii: baab010. [Epub ahead of print]2021
      Ubiquitination is an important post-translational modification, which controls protein turnover by labeling malfunctional and redundant proteins for proteasomal degradation, and also serves intriguing non-proteolytic regulatory functions. E3 ubiquitin ligases, whose substrate specificity determines the recognition of target proteins of ubiquitination, play crucial roles in ubiquitin-proteasome system. UbiNet 2.0 is an updated version of the database UbiNet. It contains 3332 experimentally verified E3-substrate interactions (ESIs) in 54 organisms and rich annotations useful for investigating the regulation of ubiquitination and the substrate specificity of E3 ligases. Based on the accumulated ESIs data, the recognition motifs in substrates for each E3 were also identified and a functional enrichment analysis was conducted on the collected substrates. To facilitate the research on ESIs with different categories of E3 ligases, UbiNet 2.0 performed strictly evidence-based classification of the E3 ligases in the database based on their mechanisms of ubiquitin transfer and substrate specificity. The platform also provides users with an interactive tool that can visualize the ubiquitination network of a group of self-defined proteins, displaying ESIs and protein-protein interactions in a graphical manner. The tool can facilitate the exploration of inner regulatory relationships mediated by ubiquitination among proteins of interest. In summary, UbiNet 2.0 is a user-friendly web-based platform that provides comprehensive as well as updated information about experimentally validated ESIs and a visualized tool for the construction of ubiquitination regulatory networks available at
  28. Autophagy. 2021 Mar 11. 1-18
      Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, fbxo22-/- mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy.Abbreviations: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.
    Keywords:  AKT1; FBXO22; KDM4B; MYC; TP53; autophagy; hormesis; ubiquitination
  29. J Proteome Res. 2021 Mar 08.
      Post-translational modifications of proteins play an important role in the regulation of cellular processes. The mass spectrometry analysis of proteome modifications offers huge potential for the study of how protein inhibitors affect the phosphosignaling mechanisms inside the cells. We have recently proposed PHONEMeS, a method that uses high-content shotgun phosphoproteomic data to build logical network models of signal perturbation flow. However, in its original implementation, PHONEMeS was computationally demanding and was only used to model signaling in a perturbation context. We have reformulated PHONEMeS as an Integer Linear Program (ILP) that is orders of magnitude more efficient than the original one. We have also expanded the scenarios that can be analyzed. PHONEMeS can model data upon perturbation on not only a known target but also deregulated pathways upstream and downstream of any set of deregulated kinases. Finally, PHONEMeS can now analyze data sets with multiple time points, which helps us to obtain better insight into the dynamics of the propagation of signals. We illustrate the value of the new approach on various data sets of medical relevance, where we shed light on signaling mechanisms and drug modes of action.
    Keywords:  cell signaling; integer linear programming; modelling; phosphoproteomics
  30. Dev Cell. 2021 Mar 02. pii: S1534-5807(21)00121-0. [Epub ahead of print]
      Beginning with the earliest studies of autophagy in cancer, there have been indications that autophagy can both promote and inhibit cancer growth and progression; autophagy regulation of organelle homeostasis is similarly complicated. In this review we discuss pro- and antitumor effects of organelle-targeted autophagy and how this contributes to several hallmarks of cancer, such as evading cell death, genomic instability, and altered metabolism. Typically, the removal of damaged or dysfunctional organelles prevents tumor development but can also aid in proliferation or drug resistance in established tumors. By better understanding how organelle-specific autophagy takes place and can be manipulated, it may be possible to go beyond the brute-force approach of trying to manipulate all autophagy in order to improve therapeutic targeting of this process in cancer.
    Keywords:  ER-phagy; autophagy; cancer; lysophagy; mitophagy
  31. J Cell Biol. 2021 May 03. pii: e202006035. [Epub ahead of print]220(5):
      Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.
  32. FASEB J. 2021 Apr;35(4): e21326
      Histone modifications play critical roles in DNA damage repair to safeguard genome integrity. However, how different histone modifiers coordinate to build appropriate chromatin context for DNA damage repair is largely unknown. Here, we report a novel interplay between the histone methyltransferase KMT5A and two E3 ligases RNF8 and RNF168 in establishing the histone modification status for DNA damage repair. KMT5A is a newly identified substrate of RNF8 in vitro and in vivo. In response to DNA double-strand breaks (DSBs), RNF8 promotes KMT5A recruitment onto damaged chromatin in a ubiquitination-dependent manner. RNF8-induced KMT5A ubiquitination increases the binding capacity of KMT5A to RNF168. Interestingly, KMT5A not only drives a local increase in H4K20 monomethylation at DSBs, but also promotes RNF168's activity in catalyzing H2A ubiquitination. We proved that the interaction between the H2A acidic patch and KMT5A R188/R189 residues is critical for KMT5A-mediated regulation of H2A ubiquitination. Taken together, our results highlight a new role for KMT5A in linking H4K20 methylation and H2A ubiquitination and provide insight into the histone modification network during DNA damage repair.
    Keywords:  DNA double-strand breaks; KMT5A; RNF168; RNF8; histone ubiquitination
  33. Immunohorizons. 2021 Mar 08. 5(3): 135-146
      The ability to modulate direct MHC class I (MHC I) Ag presentation is a desirable goal for the treatment of a variety of conditions, including autoimmune diseases, chronic viral infections, and cancers. It is therefore necessary to understand how changes in the cellular environment alter the cells' ability to present peptides to T cells. The unfolded protein response (UPR) is a signaling pathway activated by the presence of excess unfolded proteins in the endoplasmic reticulum. Previous studies have indicated that chemical induction of the UPR decreases direct MHC I Ag presentation, but the precise mechanisms are unknown. In this study, we used a variety of small molecule modulators of different UPR signaling pathways to query which UPR signaling pathways can alter Ag presentation in both murine and human cells. When signaling through the PERK pathway, and subsequent eIF2α phosphorylation, was blocked by treatment with GSK2656157, MHC I Ag presentation remain unchanged, whereas treatment with salubrinal, which has the opposite effect of GSK2656157, decreases both Ag presentation and overall cell-surface MHC I levels. Treatment with 4μ8C, an inhibitor of the IRE1α UPR activation pathway that blocks splicing of Xbp1 mRNA, also diminished MHC I Ag presentation. However, 4μ8C treatment unexpectedly led to an increase in eIF2α phosphorylation in addition to blocking IRE1α signaling. Given that salubrinal and 4μ8C lead to eIF2α phosphorylation and similar decreases in Ag presentation, we conclude that UPR signaling through PERK, leading to eIF2α phosphorylation, results in a modest decrease in direct MHC I Ag presentation.
  34. Nat Chem Biol. 2021 Mar 11.
      Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.
  35. Hum Mol Genet. 2021 Mar 09. pii: ddab040. [Epub ahead of print]
      Mutations in the WFS1 gene, encoding wolframin (WFS1), cause endoplasmic reticulum (ER) stress and are associated with a rare autosomal recessive disorder known as Wolfram syndrome (WS). WS is clinically characterized by childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus, and neurological signs. We identified two novel WFS1 mutations in a patient with WS, namely, c.316-1G > A (in intron 3) and c.757A > T (in exon 7). Both mutations, located in the N-terminal region of the protein, were predicted to generate a truncated and inactive form of WFS1. We found that although the WFS1 protein was not expressed in peripheral blood mononuclear cells (PBMCs) of the proband, no constitutive ER stress activation could be detected in those cells. In contrast, WS proband's PBMCs produced very high levels of pro-inflammatory cytokines (i.e. TNF-α, IL-1β, and IL-6) in the absence of any stimulus. WFS1 silencing in PBMCs from control subjects by means of small RNA interference also induced a pronounced pro-inflammatory cytokine profile. The same cytokines were also significantly higher in sera from the WS patient as compared to matched healthy controls. Moreover, the chronic inflammatory state was associated with a dominance of pro-inflammatory T helper 17 (Th17)-type cells over regulatory T (Treg) lymphocytes in the WS PBMCs. The identification of a state of systemic chronic inflammation associated with WFS1 deficiency may pave the way to innovative and personalized therapeutic interventions in WS.
  36. Nat Chem Biol. 2021 Mar 08.
      O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.
  37. Trends Cell Biol. 2021 Mar 05. pii: S0962-8924(21)00024-6. [Epub ahead of print]
      Ubiquitin and ubiquitin-like proteins (UBLs) function as critical post-translational modifiers in the maintenance of genome stability. Ubiquitin/UBL-conjugating enzymes (E2s) are responsible, as part of a wider enzymatic cascade, for transferring single moieties or polychains of ubiquitin/UBLs to one or multiple residues on substrate proteins. Recent advances in structural and mechanistic understanding of how ubiquitin/UBL substrate attachment is orchestrated indicate that E2s can exert control over chain topology, substrate-site specificity, and downstream physiological effects to help maintain genome stability. Drug discovery efforts have typically focussed on modulating other members of the ubiquitin/UBL cascades or the ubiquitin-proteasome system. Here, we review the current standing of E2s in genome stability and revisit their potential as pharmacological targets for developing novel anti-cancer therapies.
    Keywords:  DNA repair; E2-conjugating enzymes (E2s); cell cycle; genome stability; telomeres; ubiquitin and ubiquitin-like proteins (UBLs)
  38. Cell Death Dis. 2021 Mar 12. 12(3): 265
      NOXA, a BH3-only proapoptotic protein involved in regulating cell death decisions, is highly expressed but short-lived in colorectal cancer (CRC). Neddylated cullin-5 (CUL5)-mediated ubiquitination and degradation of NOXA is crucial to prevent its overaccumulation and maintain an appropriate action time. However, how this process is manipulated by CRC cells commonly exposed to oxidative stress remain unknown. The peroxiredoxin PRDX1, a conceivable antioxidant overexpressed in CRC tissues, has been shown to inhibit apoptosis and TRAF6 ubiquitin-ligase activity. In this study, we found that PRDX1 inhibits CRC cell apoptosis by downregulating NOXA. Mechanistically, PRDX1 promotes NOXA ubiquitination and degradation, which completely depend on CUL5 neddylation. Further studies have demonstrated that PRDX1 oligomers bind with both the Nedd8-conjugating enzyme UBE2F and CUL5 and that this tricomplex is critical for CUL5 neddylation, since silencing PRDX1 or inhibiting PRDX1 oligomerization greatly dampens CUL5 neddylation and NOXA degradation. An increase in reactive oxygen species (ROS) is not only a hallmark of cancer cells but also the leading driving force for PRDX1 oligomerization. As shown in our study, although ROS play a role in upregulating NOXA mRNA transcription, ROS scavenging in CRC cells by N-acetyl-L-cysteine (NAC) can significantly reduce CUL5 neddylation and extend the NOXA protein half-life. Therefore, in CRC, PRDX1 plays a key role in maintaining intracellular homeostasis under conditions of high metabolic activity by reinforcing UBE2F-CUL5-mediated degradation of NOXA, which is also evidenced in the resistance of CRC cells to etoposide treatment. Based on these findings, targeting PRDX1 could be an effective strategy to overcome the resistance of CRC to DNA damage-inducing chemotherapeutics.