bims-proteo Biomed News
on Proteostasis
Issue of 2020‒06‒21
forty papers selected by
Eric Chevet

  1. FEBS Lett. 2020 Jun 15.
    Park WJ, Park JW.
      The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism, and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER-associated-protein-degradation, and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long-chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis, and cell-cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events.
    Keywords:  Sphingolipid; acyl chain length; ceramide; disease; endoplasmic reticulum stress
  2. Sci Adv. 2020 Jun;6(23): eaba0745
    Kastan JP, Dobrikova EY, Bryant JD, Gromeier M.
      Eukaryotic protein synthesis control at multiple levels allows for dynamic, selective responses to diverse conditions, but spatial organization of translation initiation machinery as a regulatory principle has remained largely unexplored. Here we report on a role of constitutive repressor of eIF2α phosphorylation (CReP) in translation of poliovirus and the endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP) at the ER. Functional, proximity-dependent labeling and cell fractionation studies revealed that CReP, through binding eIF2α, anchors translation initiation machinery at the ER and enables local protein synthesis in this compartment. This ER site was protected from the suppression of cytoplasmic protein synthesis by acute stress responses, e.g., phosphorylation of eIF2α(S51) or mTOR blockade. We propose that partitioning of translation initiation machinery at the ER enables cells to maintain active translation during stress conditions associated with global protein synthesis suppression.
  3. Cytoskeleton (Hoboken). 2020 Jun 15.
    Zhang D.
      Eukaryotic membrane-bound organelles, exhibiting distinctive morphologies, dynamics and functions, are interconnected at membrane contact sites (MCSs) through numerous tethering machineries. MCSs are required for many fundamental cellular processes, such as non-vesicular lipid transfer, calcium transport and organelle homeostasis. Actin cytoskeleton and myosin motors are known to dynamically interact with different membrane boundaries, facilitating organelle movements and partitioning. Intriguingly, recent studies have pinpointed a special participation of actomyosin at various MCSs involving the endoplasmic reticulum (ER), the most extensive membranous organelle in the cell. Here, I summarize emerging roles of ER MCSs in modulating actomyosin structures and discuss feedback functions of such actomyosin regulation at these MCSs. This article is protected by copyright. All rights reserved.
    Keywords:  Actomyosin dynamics; ER contacts remodeling; ER-associated organelle fission; Membrane contact sites
  4. Nat Microbiol. 2020 Jun 15.
    Vallejo-Gracia A, Chen IP, Perrone R, Besnard E, Boehm D, Battivelli E, Tezil T, Krey K, Raymond KA, Hull PA, Walter M, Habrylo I, Cruz A, Deeks S, Pillai S, Verdin E, Ott M.
      Quiescence is a hallmark of CD4+ T cells latently infected with human immunodeficiency virus 1 (HIV-1). While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that, in resting T cells, FOXO1 inhibition impaired autophagy and induced endoplasmic reticulum (ER) stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of protein kinase R-like ER kinase, an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link of FOXO1, ER stress and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.
  5. Autophagy. 2020 Jun 19.
    Mookherjee D, Das S, Mukherjee R, Bera M, Jana SC, Chakrabarti S, Chakrabarti O.
      Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate "mitoplasts". This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The "mitoplasts" generated are degraded by reticulo-mito-phagy - simultaneously affecting dual organelle turnover.
    Keywords:  AMFR/GP78; OPA1; RETREG1/FAM134B; autophagy; mitoplast; reticulo-mito-phagy
  6. J Pharmacol Exp Ther. 2020 Jun 17. pii: jpet.119.264481. [Epub ahead of print]
    Forno F, Maatuf Y, Boukeileh S, Dipta P, Mahameed M, Darawshi O, Ferreira V, Rada P, Garcia-Martinez I, Gross E, Priel A, Martinez Valverde A, Tirosh B.
      Schizophrenia is a mental disease that results in decreased life expectancy and wellbeing, by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. While the second generation of antipsychotics (SGA), Olanzapine and Aripiprazole are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain as well as dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We, therefore, investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole, not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of IRE1 and PERK, two major transducers of the UPR. Cells underwent apoptosis upon Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2 cells protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The anti-schizophrenic drug Aripiprazole has cytotoxic properties at high concentrations. This study shows that cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in died-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism for the metabolic derangement associated with anti-schizophrenic drugs.
    Keywords:  Ca imaging; cell death; drug toxicity; glutathione; pharmacodynamics; protein kinases
  7. Front Cell Dev Biol. 2020 ;8 343
    Morel E.
      Autophagy mobilizes a variety of intracellular endomembranes to ensure a proper stress response and the maintenance of cellular homeostasis. While the process of de novo biogenesis of pre-autophagic structures is not yet fully characterized, the role of the endoplasmic reticulum (ER) appears to be crucial in early steps of autophagic process. Here, I review and discuss various aspects of ER and ER-driven membrane contact site requirements and effects on mammalian organelles and endomembrane biogenesis, in particular during the early steps of autophagy-related membrane dynamics.
    Keywords:  ER; autophogosome; biogenesis; lipids; membrane contact site
  8. Trends Cancer. 2020 Jun 12. pii: S2405-8033(20)30167-9. [Epub ahead of print]
    Khateb A, Ronai ZA.
      Understanding genetic and epigenetic changes that underlie abnormal proliferation of hematopoietic stem and progenitor cells is critical for development of new approaches to monitor and treat leukemia. The unfolded protein response (UPR) is a conserved adaptive signaling pathway that governs protein folding, secretion, and energy production and serves to maintain protein homeostasis in various cellular compartments. Deregulated UPR signaling, which often occurs in hematopoietic stem cells and leukemia, defines the degree of cellular toxicity and perturbs protein homeostasis, and at the same time, offers a novel therapeutic target. Here, we review current knowledge related to altered UPR signaling in leukemia and highlight possible strategies for exploiting the UPR as treatment for this disease.
    Keywords:  endoplasmic reticulum stress; hematological malignancy; hematopoietic stem cell; lymphoid leukemia; myeloid leukemia; unfolded protein response
  9. Eur J Pharmacol. 2020 Jun 16. pii: S0014-2999(20)30380-0. [Epub ahead of print] 173288
    Sureda A, Alizadeh J, Nabavi SF, Berindan Neagoe I, Cismaru CA, Jeandet P, Los MJ, Clementi E, Nabavi SM, Ghavami S.
      In December 2019, many pneumonia cases with unidentified sources appeared in Wuhan, Hubei, China, with clinical symptoms like viral pneumonia. Deep sequencing analysis of samples from lower respiratory tract revealed a novel coronavirus, called 2019 novel coronavirus (2019-nCoV). Currently there is a rapid global spread. World Health Organization declare the disease a pandemic condition. The pathologic source of this disease was a new RNA virus from Coronaviridae family, which was named COVID-19. SARS-CoV-2 entry starts with the binding of the spike glycoprotein expressed on the viral envelope to ACE2 on the alveolar surface followed by clathrin-dependent endocytosis of the SARS-CoV-2 and ACE2 complex. SARS-CoV-2 enters the cells through endocytosis process, which is possibly facilitated, via a pH dependent endosomal cysteine protease cathepsins. Once inside the cells, SARS-CoV-2 exploits the endogenous transcriptional machinery of alveolar cells to replicate and spread through the entire lung. Endosomal acidic pH for SARS-CoV-2 processing and internalization is critical. After entering the cells, it possibly activates or hijack many intracellular pathways in favor of its replication. In the current opinion article, we will explain the possible involvement of unfolded protein response as a cellular stress response to the SARS-CoV-2 infection.
    Keywords:  Endoplasmic reticulum; IRE1; PERK; Spliced XBP1; Unfolded protein response
  10. Cell Struct Funct. 2020 Jun 18.
    Matsui Y, Hirata Y, Wada I, Hosokawa N.
      Collagen is the most abundant protein in animal tissues and is critical for their proper organization. Nascent procollagens in the endoplasmic reticulum (ER) are considered too large to be loaded into coat protein complex II (COPII) vesicles, which have a diameter of 60-80 nm, for exit from the ER and transport to the Golgi complex. To study the transport mechanism of procollagen IV, which generates basement membranes, we introduced a cysteine-free GFP tag at the N-terminus of the triple helical region of the α1(IV) chain (cfSGFP2-col4a1), and examined the dynamics of this protein in HT-1080 cells, which produce endogenous collagen IV. cfSGFP2-col4a1 was transported from the ER to the Golgi by vesicles, which were a similar size as small cargo carriers. However, mCherry-ERGIC53 was recruited to α1-antitrypsin-containing vesicles, but not to cfSGFP2-col4a1-containing vesicles. Knockdown analysis revealed that Sar1 and SLY1/SCFD1 were required for transport of cfSGFP2-col4a1. TANGO1, CUL3, and KLHL12 were not necessary for the ER-to-Golgi trafficking of procollagen IV. Our data suggest that procollagen IV is exported from the ER via an enlarged COPII coat carrier and is transported to the Golgi by unique transport vesicles without recruitment of ER-Golgi intermediate compartment membranes. Key words: collagen, procollagen IV, endoplasmic reticulum, ER-to-Golgi transport, ERGIC.
    Keywords:  ER-to-Golgi transport; ERGIC; collagen; endoplasmic reticulum; procollagen IV
  11. Biol Open. 2020 Jun 17. pii: bio.046391. [Epub ahead of print]
    Nüske E, Marini G, Richter D, Leng W, Bogdanova A, Franzmann TM, Pigino G, Alberti S.
      Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants of eIF2B suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament formation is an efficient mechanism that allows yeast cells to adapt to fluctuating environments.
    Keywords:  Budding yeast; Protein assembly; Regulation of translation; Starvation; Stress response
  12. J Cell Biol. 2020 Jul 06. pii: e201908087. [Epub ahead of print]219(7):
    Tang D, Sandoval W, Lam C, Haley B, Liu P, Xue D, Roy D, Patapoff T, Louie S, Snedecor B, Misaghi S.
      Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.
  13. IUBMB Life. 2020 Jun 13.
    Liu TW, Liu F, Kang J.
      The endogenous non-coding microRNA (miRNA) let-7b-5p is highly expressed in the blood of patients with acute pulmonary embolism (PE). However, the mechanism underlying the involvement of let-7b-5p in acute PE remains unclear. To address this, we investigated the role of let-7b-5p in acute PE in both in vitro and in vivo experimental models. The results showed that let-7b-5p upregulated the expression of stress-associated endoplasmic reticulum protein 1 (SERP1) at the post-transcriptional level. SERP1 activation leads to modulation of its chaperone protein SEC61B in the response of endoplasmic reticulum (ER) stress. Furthermore, our data show that the unfolded protein response was triggered and activation of unfolded proteins GRP78, PERK, RNF121, and CHOP occurred through the PERK-CHOP pathway, resulting in an inflammatory response and apoptosis of lung epithelial cells. These characteristics were promoted by the in vitro expression of a let-7b-5p mimic; conversely, transfection with a let-7b-5p inhibitor decreased the response of ER stress in acute PE. The results from this study thus provide evidence that let-7b-5p promotes protein processing during ER stress response by upregulating SERP1 expression, ultimately resulting in an inflammatory response and apoptosis of lung cells, cumulatively playing a critical role in the pathogenesis of acute PE.
    Keywords:  acute pulmonary embolism; endoplasmic reticulum stress; inositol-requiring kinase-I; let-7b-5p; stress-associated endoplasmic reticulum protein 1 (SERP1)
  14. Trends Cell Biol. 2020 Jun 16. pii: S0962-8924(20)30101-X. [Epub ahead of print]
    Bhattarai KR, Chaudhary M, Kim HR, Chae HJ.
      Recent work provides evidence for the new terminology, 'endoplasmic reticulum (ER) stress response or sensing failure', in relation to metabolic disease. We seek to identify and amass possible conditions of ER stress response failure in various metabolic and age-related pathogenesis, including obesity and diabetes.
    Keywords:  ER proteostasis; ER stress; ER stress response failure; aging; metabolic diseases; sXBP1
  15. Cell Death Differ. 2020 Jun 17.
    Li W, Wang Q, Qi X, Lu H, Chen Y, Shi J, Wang F, Wang Z, Lu Z, Yan Q, Wang C, Gao SJ, Lu C.
      Kaposi's sarcoma (KS), a highly angiogenic and invasive vascular tumor, is the most common AIDS-associated cancer caused by KS-associated herpesvirus (KSHV) infection. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) contributes to KSHV-induced cell motility (PLoS Pathog. 15:e1007578, 2019). However, the role of vIRF1 in KSHV-induced angiogenesis remains unknown. Here, using two in vivo angiogenesis models including the chick chorioallantoic membrane assay (CAM) and the matrigel plug angiogenesis assay in mice, we show that vIRF1 promotes angiogenesis by upregulating CUB domain (for complement C1r/C1s, Uegf, Bmp1) containing protein 1 (CDCP1). Mechanistically, vIRF1 enhances the expression of transcription factor lymphoid enhancer-binding factor 1 (Lef1) and binds to Lef1 to promote CDCP1 transcription. Meanwhile, vIRF1 degrades metastasis suppressor CD82 through an ubiquitin-proteasome pathway by recruiting E3 ubiquitin ligase AMFR to CD82, which protects CDCP1 from CD82-mediated, palmitoylation-dependent degradation. CDCP1 activates AKT signaling, which is required for vIRF1-induced cell motility but not angiogenesis. Our results illustrate that, by hijacking Lef1 and CD82, vIRF1 upregulates CDCP1 to promote angiogenesis and cell invasion. These novel findings demonstrate the vIRF1 targets multiple cellular proteins and pathways to promote the pathogenesis of KS, which could be attractive therapeutic targets for KSHV-induced malignancies.
  16. Nat Immunol. 2020 Jun 15.
    Jia M, Qin D, Zhao C, Chai L, Yu Z, Wang W, Tong L, Lv L, Wang Y, Rehwinkel J, Yu J, Zhao W.
      Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.
  17. Autophagy. 2020 Jun 19.
    Mesquita A, Glenn J, Jenny A.
      As one of the major, highly conserved catabolic pathways, autophagy delivers cytosolic components to lysosomes for degradation. It is essential for development, cellular homeostasis, and coping with stress. Reduced autophagy increases susceptibility to protein aggregation diseases and leads to phenotypes associated with aging. Of the three major forms of autophagy, macroautophagy (MA) can degrade organelles or aggregated proteins, and chaperone-mediated autophagy is specific for soluble proteins containing KFERQ-related targeting motifs. During endosomal microautophagy (eMI), cytoplasmic proteins are engulfed into late endosomes in an ESCRT machinery-dependent manner. eMI can be KFERQ-specific or occur in bulk and be induced by prolonged starvation. Its physiological regulation and function, however, are not understood. Here, we show that eMI in the Drosophila fat body, akin to the mammalian liver, is induced upon oxidative or genotoxic stress in an ESCRT and partially Hsc70-4-dependent manner. Interestingly, eMI activation is selective, as ER stress fails to elicit a response. Intriguingly, we find that reducing MA leads to a compensatory enhancement of eMI, suggesting a tight interplay between these degradative processes. Furthermore, we show that mutations in DNA damage response genes are sufficient to trigger eMI and that the response to oxidative stress is under the control of MAPK/JNK signaling. Our data suggest that, controlled by various signaling pathways, eMI allows an organ to react and adapt to specific types of stress and is thus likely critical to prevent disease.
    Keywords:  DNA damage; ER stress; ROS; autophagy; microautophagy; oxidative stress; proteostasis
  18. Curr Top Med Chem. 2020 Jun 18.
    Kabir MT, Uddin MS, Abdeen A, Ashraf GM, Perveen A, Hafeez A, Bin-Jumah MN, Abdel-Daim MM.
      Several proteolytic systems including ubiquitin (Ub)-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy are used by the mammalian cells to remove misfolded proteins (MPs). UPS mediates degradation of most of the MPs, where Ub-conjugated substrates are deubiquitinated, unfolded, and pass through the proteasome's narrow chamber, and eventually break into smaller peptides. It has been observed that the substrates that show a specific degradation signal, the KFERQ sequence motif, can be delivered to and go through CMA-mediated degradation in lysosomes. Macroautophagy can help in the degradation of substrates that are prone to aggregation and resistant to both the CMA and UPS. In the aforesaid case, cargoes are separated into autophagosomes before lysosomal hydrolase-mediated degradation. Even though the majority of the aggregated and MPs in the human proteome can be removed via cellular protein quality control (PQC), some mutant and native proteins tend to aggregate into β-sheet-rich oligomers that exhibit resistance to all identified proteolytic processes and can, therefore, grow into extracellular plaques or inclusion bodies. Indeed, the buildup of protease-resistant aggregated and MPs is a usual process underlying various protein misfolding disorders, including neurodegenerative diseases (NDs) for example Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. In this article, we have focused on the contribution of PQC in the degradation of pathogenic proteins in NDs.
    Keywords:  Protein misfolding; amyloid β; chaperone mediated autophagy; macroautophagy; neurodegeneration; tau.; ubiquitin-proteasome system
  19. Cell Stress. 2020 Apr 15. 4(6): 151-153
    Lee SH, Hadipour-Lakmehsari S, Gramolini AO.
      The sarco-endoplasmic reticulum (SR/ER) is the largest membrane-bound organelle in eukaryotic cells and plays important roles in essential cellular processes, and in development and progression of many cardiac diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. In a recently published study led by Lee et al. (Nat Commun 11(1):965), we reported a cardiac enriched SR/ER membrane protein REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes resulted in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction to heart failure induction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrated cardiac dysfunction with dilated cardiac chambers, increased cardiac fibrosis, and reduced ejection fraction. These results demonstrate the critical role of REEP5 in SR/ER organization and function.
    Keywords:  SR organization; cardiac ER stress; cardiac myocytes; heart development; heart function; zebrafish
  20. J Cell Sci. 2020 Jun 16. pii: jcs.240085. [Epub ahead of print]
    Schregle R, Mueller S, Legler DF, Rossy J, Krueger WA, Groettrup M.
      Dendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the ubiquitin-like modifier FAT10 that localises to aggresomes that are structures similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here we have investigated whether FAT10 is involved in the formation and turnover of DALIS and whether proteins accumulating in DALIS can be FAT10ylated. We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing which may provide a functional rationale why FAT10 is selectively induced upon DC maturation.
    Keywords:  DALIS; Dendritic cells; FAT10; Proteasome; Ubiquitin
  21. J Infect. 2020 Jun 11. pii: S0163-4453(20)30398-4. [Epub ahead of print]
    Ha DP, Van Krieken R, Carlos A, Lee AS.
    Keywords:  Coronavirus; Endoplasmic reticulum; GRP78; Host factor; Viral infection
  22. J Cell Biol. 2020 Aug 03. pii: e201910119. [Epub ahead of print]219(8):
    Lu MS, Drubin DG.
      Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.
  23. Cells. 2020 Jun 16. pii: E1468. [Epub ahead of print]9(6):
    Sanwald JL, Poschmann G, Stühler K, Behrends C, Hoffmann S, Willbold D.
      The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP-which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells-we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP's proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.
    Keywords:  APEX2; ATG8; GABARAP; autophagy; exosome; extracellular vesicle; proximity; secretion
  24. Int J Mol Sci. 2020 Jun 17. pii: E4318. [Epub ahead of print]21(12):
    Rao L, Xu Y, Reineke LC, Bhattacharya A, Tyryshkin A, Shin JN, Eissa NT.
      Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.
    Keywords:  MG132; alpha one antitrypsin; alpha one antitrypsin deficiency; induced pluripotent stem cells; proteasome inhibitor; stress granule
  25. Autophagy. 2020 Jun 16.
    Chu Y, Kang Y, Yan C, Yang C, Zhang T, Huo H, Liu Y.
      Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a de-ubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. RNF31 knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. OTULIN knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In OTULIN knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.
    Keywords:  ATG13; LUBAC; OTULIN; RNF31; autophagosome; autophagy; linear ubiquitination
  26. Proc Natl Acad Sci U S A. 2020 Jun 15. pii: 201921554. [Epub ahead of print]
    Graves JD, Lee YJ, Liu K, Li G, Lin FT, Lin WC.
      Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.
    Keywords:  E2F1; SENP3; SUMO2; oxidative stress; sumoylation
  27. Cell Biochem Biophys. 2020 Jun 19.
    Liu Y, Jin M, Gao Y, Wang Y, Xue S, Wang L, Xuan C.
      The ubiquitin proteasome pathway is conserved from yeast to mammals and is necessary for the targeted degradation of most short-lived proteins in eukaryotic cells. Its protein substrates include cell cycle regulatory proteins and proteins that are not properly folded in the endoplasmic reticulum. Owing to the ubiquity of its protein substrates, ubiquitination regulates a variety of cellular activities, including cell proliferation, apoptosis, autophagy, endocytosis, DNA damage repair, and immune response. With new genomic data continuously being obtained, ubiquitination through genomic data analysis will be an effective method. We obtained 83 overlapping genes from four glioma databases, which differed from ubiquitin ligase Nrdp1 expression, including 36 downregulated and 47 upregulated genes. The KEGG pathways, molecular functions, cellular components, and biological processes potentially associated with Nrdp1 were obtained using GSEA and Cytoscape. In human gliomas, differences in the expression of Nrdp1 were identified between nontumor brain tissue and different glioma tissues, but no difference in expression was found between low‑grade glioma (LGG) and anaplastic glioma (AG). In survival analysis, we found no significant association between Nrdp1 expression level and patient prognosis.
    Keywords:  Bioinformatics; GSEA (gene set enrichment analysis); Genomics; Glioma; Nrdp1 (neuregulin receptor degradation protein-1); Ubiquitination
  28. Am J Respir Cell Mol Biol. 2020 Jun 18.
    Jiang D, Cui H, Xie N, Banerjee S, Liu R, Dai H, Thannickal VJ, Liu G.
      While UPRER is well known, UPRmt has not been recognized, in alveolar epithelial cells. Furthermore, ER stress and mitochondrial dysfunction are frequently encountered in alveolar epithelial cells from an array of lung disorders. However, these two scenarios have been often regarded as separate mechanisms contributing to the pathogeneses. It is unclear if there is interplay between these two phenomena or an integrator that couples these two signaling cascades in the stressed alveolar epithelial cells from those pathologies. In this study, we defined UPRmt in alveolar epithelial cells and identified ATF4, but not ATF5, as the key regulator of UPRmt. We found that UPRER led to UPRmt and mitochondrial dysfunction in an ATF4 dependent manner. In contrast, mitochondrial stresses did not activate UPRER. We found that alveolar epithelial ATF4 and UPRmt were induced in aged mice with experimental pulmonary fibrosis as well as in IPF patients. Finally, we found that inducible expression of ATF4 in mouse alveolar epithelial cells aggravated pulmonary UPRmt, lung inflammation, body weight loss and death upon bleomycin induced lung injury. In conclusion, ER stress induces ATF4-dependent UPRmt and mitochondrial dysfunction, indicating a novel mechanism by which ER stress contributes to the pathogeneses of a variety of pulmonary disorders.
  29. Int J Mol Sci. 2020 Jun 14. pii: E4237. [Epub ahead of print]21(12):
    Kanuka M, Ouchi F, Kato N, Katsuki R, Ito S, Miura K, Hikida M, Tamura T.
      Spinocerebellar ataxia (SCA) is one of the most severe neurodegenerative diseases and is often associated with misfolded protein aggregates derived from the genetic mutation of related genes. Recently, mutations in CD10 such as C143Y have been identified as SCA type 43. CD10, also known as neprilysin or neuroendopeptidase, digests functional neuropeptides, such as amyloid beta, in the extracellular region. In this study, we explored the cellular behavior of CD10 C143Y to gain an insight into the functional relationship of the mutation and SCA pathology. We found that wild-type CD10 is expressed on the plasma membrane and exhibits endopeptidase activity in a cultured cell line. CD10 C143Y, however, forms a disulfide bond-mediated oligomer that does not appear by the wild-type CD10. Furthermore, the CD10 C143Y mutant was retained in the endoplasmic reticulum (ER) by the molecular chaperone BiP and was degraded through the ER-associated degradation (ERAD) process, in which representative ERAD factors including EDEM1, SEL1L, and Hrd1 participate in the degradation. Suppression of CD10 C143Y ERAD recovers intracellular transport but not enzymatic activity. Our results indicate that the C143Y mutation in CD10 negatively affects protein maturation and results in ER retention and following ERAD. These findings provide beneficial insight into SCA type 43 pathology.
    Keywords:  CD10; ER quality control; disulfide bond; endoplasmic reticulum-associated degradation (ERAD); spinocerebellar ataxia
  30. Autophagy. 2020 Jun 16. 1-2
    Wen X, Klionsky DJ.
      There is a type of noncanonical autophagy, which is independent of ATG5 (autophagy related 5), also referred to as alternative autophagy. Both canonical and ATG5-independent alternative autophagy require the initiator ULK1 (unc-51 like kinase 1), but how ULK1 regulates these two types of autophagy differently remains unclear. A recent paper from Torii et al. demonstrates that phosphorylation of ULK1 at Ser746 by RIPK3 (receptor interacting serine/threonine kinase 3) is the key difference between these two types of autophagy; this phosphorylation is exclusively found during alternative autophagy.
    Keywords:  Alternative autophagy; Golgi; RIPK3; ULK1; phosphorylation
  31. J Cell Sci. 2020 Jun 16. pii: jcs.241356. [Epub ahead of print]
    Tamir TY, Bowman BM, Agajanian MJ, Goldfarb D, Schrank TP, Stohrer T, Hale AE, Siesser PF, Weir SJ, Murphy RM, LaPak KM, Weissman BE, Moorman NJ, Major MB.
      NFE2L2/NRF2 is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, and conversely NRF2 activity is diminished with age and in neurodegenerative as well as metabolic disorders. Though NRF2 activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here we used a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the understudied protein kinase Brain Specific Kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives AMPK signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppressed ribosome-RNA complexes, global protein synthesis, and NRF2 protein levels. Collectively our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis may prove useful for therapeutically targeting NRF2 in human disease.
    Keywords:  AMPK; BRSK1; BRSK2; Functional genomics; Kinase; MTOR; NRF2; Oxidative stress response; Phosphoproteomics
  32. Proc Natl Acad Sci U S A. 2020 Jun 15. pii: 201920109. [Epub ahead of print]
    Castanzo DT, LaFrance B, Martin A.
      Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.
    Keywords:  AAA+ ATPase; Msp1; Pex15; Pex3
  33. Int J Mol Sci. 2020 Jun 12. pii: E4196. [Epub ahead of print]21(12):
    Csizmadia T, Lőw P.
      Deubiquitinating enzymes (DUBs) have an essential role in several cell biological processes via removing the various ubiquitin patterns as posttranslational modification forms from the target proteins. These enzymes also contribute to the normal cytoplasmic ubiquitin pool during the recycling of this molecule. Autophagy, a summary name of the lysosome dependent self-degradative processes, is necessary for maintaining normal cellular homeostatic equilibrium. Numerous forms of autophagy are known depending on how the cellular self-material is delivered into the lysosomal lumen. In this review we focus on the colorful role of DUBs in autophagic processes and discuss the mechanistic contribution of these molecules to normal cellular homeostasis via the possible regulation forms of autophagic mechanisms.
    Keywords:  DUB; cargo degradation; lysosome; ubiquitin; vesicle fusion
  34. Cell Chem Biol. 2020 Jun 18. pii: S2451-9456(20)30193-8. [Epub ahead of print]27(6): 637-639
    Au YZ, Wang T, Sigua LH, Qi J.
      Qu et al. (2020) demonstrate a peptide-induced targeted degradation of the alpha-synuclein protein, a hallmark of Parkinson's disease. Using a modular three-component design, the target-protein-specific, cell-permeable peptide disposed of alpha-synuclein via the ubiquitin-proteasome pathway rather than the standard autophagy-lysosome pathway.
  35. Biomolecules. 2020 Jun 14. pii: E903. [Epub ahead of print]10(6):
    Leboeuf D, Pyatkov M, Zatsepin TS, Piatkov K.
      Recognition of danger signals by a cell initiates a powerful cascade of events generally leading to inflammation. Inflammatory caspases and several other proteases become activated and subsequently cleave their target proinflammatory mediators. The irreversible nature of this process implies that the newly generated proinflammatory fragments need to be sequestered, inhibited, or degraded in order to cancel the proinflammatory program or prevent chronic inflammation. The Arg/N-degron pathway is a ubiquitin-dependent proteolytic pathway that specifically degrades protein fragments bearing N-degrons, or destabilizing residues, which are recognized by the E3 ligases of the pathway. Here, we report that the Arg/N-degron pathway selectively degrades a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. Partial ablation of the Arg/N-degron pathway greatly increases IL-1β secretion, indicating the importance of this ubiquitous pathway in the initiation and resolution of inflammation. Thus, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and as an "off switch" for inflammatory responses through the selective degradation of proinflammatory fragments.
    Keywords:  Arg/N-degron pathway; UBR-ubiquitin ligases; inflammation; inflammatory caspases; proteolysis; ubiquitin
  36. Cell Rep. 2020 Jun 16. pii: S2211-1247(20)30752-X. [Epub ahead of print]31(11): 107772
    Swaim CD, Canadeo LA, Monte KJ, Khanna S, Lenschow DJ, Huibregtse JM.
      ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-β-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.
    Keywords:  ERVE Nairovirus vOTU; ISG15; LFA-1; SARS-CoV-2/COVID-19 PL(pro); TLRs; foot and mouth disease virus Lb(pro); influenza B NS1; interferon-α/β; interferon-γ
  37. Cancers (Basel). 2020 Jun 16. pii: E1595. [Epub ahead of print]12(6):
    Gatti V, Bernassola F, Talora C, Melino G, Peschiaroli A.
      The ubiquitin system is a dynamic regulatory pathway controlling the activity, subcellular localization and stability of a myriad of cellular proteins, which in turn affects cellular homeostasis through the regulation of a variety of signaling cascades. Aberrant activity of key components of the ubiquitin system has been functionally linked with numerous human diseases including the initiation and progression of human tumors. In this review, we will contextualize the importance of the two main components of the ubiquitin system, the E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs), in the etiology of squamous cell carcinomas (SCCs). We will discuss the signaling pathways regulated by these enzymes, emphasizing the genetic and molecular determinants underlying their deregulation in SCCs.
    Keywords:  E3 ubiquitin ligase; deubiquitinating enzymes; oncogenes; squamous cell carcinoma; tumor suppressor
  38. J Mol Cell Cardiol. 2020 Jun 14. pii: S0022-2828(20)30213-3. [Epub ahead of print]
    Qi L, Zang H, Wu W, Nagarkatti P, Nagarkatti M, Liu Q, Robbins J, Wang X, Cui T.
      Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.
    Keywords:  Autolysosome efflux; CYLD; Cardiomyocytes; Deubiquitinating enzymes; Pressure overload
  39. Nat Struct Mol Biol. 2020 Jun 15.
    Wu T, Yoon H, Xiong Y, Dixon-Clarke SE, Nowak RP, Fischer ES.
      Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems offer unprecedented temporal and spatial control over protein function. Here, we review recent developments in this field, with a particular focus on the use of degraders as research tools to interrogate complex biological problems.