bims-proteo Biomed News
on Proteostasis
Issue of 2022‒03‒06
thirty papers selected by
Eric Chevet

  1. Cell Mol Life Sci. 2022 Mar 01. 79(3): 167
      The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquitinated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorders.
    Keywords:  Lysosome; Protein homeostasis network; Protein stability; Regeneration; Reprogramming; Stress response
  2. Autophagy. 2022 Feb 27. 1-2
      The endoplasmic reticulum (ER) carries out essential cellular functions ranging from protein trafficking to metabolite signaling. ER function is maintained in part by quality control pathways including ER degradation by selective autophagy (reticulophagy) during conditions of cellular stress. Reticulophagy is known to be important for cellular responses to starvation and protein folding stress, but no natural role during development had been identified. While investigating ER remodeling during the conserved cell differentiation process of meiosis in budding yeast, we unexpectedly observed developmentally regulated reticulophagy that was driven by expression of the autophagy receptor Atg40. This reticulophagy was coordinated with massive morphological rearrangement of the ER, including movement of most cortical ER away from the cell periphery. As meiotic reticulophagy prevents specific ER subpopulations from being inherited by gametes, we propose that it serves a quality control role, preventing deleterious material from being passed on to subsequent generations.
    Keywords:  Atg40; ERphagy; endoplasmic reticulum; gametes; meiosis; quality control; reticulophagy
  3. J Exp Bot. 2022 Mar 05. pii: erac002. [Epub ahead of print]
      Physiological effects mediated by melatonin are attributable to its potent antioxidant activity as well as its role as a signaling molecule in inducing a vast array of melatonin-mediated genes. Here, we propose melatonin as a signaling molecule essential for protein quality control (PQC) in plants. PQC occurs by the coordinated activities of three systems: the chaperone network, autophagy, and the ubiquitin-proteasome system. With regard to the melatonin-mediated chaperone pathway, melatonin increases thermotolerance by induction of heat shock proteins and confers endoplasmic reticulum stress tolerance by increasing endoplasmic reticulum chaperone proteins. In chloroplasts, melatonin-induced chaperones, including Clps and CpHSP70s, play key roles in the PQC of chloroplast-localized proteins, such as Lhcb1, Lhcb4, and RBCL, during growth. Melatonin regulates PQC by autophagy processes, in which melatonin induces many autophagy (ATG) genes and autophagosome formation under stress conditions. Finally, melatonin-mediated plant stress tolerance is associated with up-regulation of stress-induced transcription factors, which are regulated by the ubiquitin-proteasome system. In this review, we propose that melatonin plays a pivotal role in PQC and consequently functions as a pleiotropic molecule under non-stress and adverse conditions in plants.
    Keywords:  AAA+ family of ATPase caseinolytic proteases; autophagosome; endoplasmic reticulum stress; heat shock proteins; melatonin; mitogen-activated protein kinase; ubiquitin ligases; unfolded protein response
  4. Autophagy. 2022 Feb 28. 1-14
      Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4',6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell.
    Keywords:  ATG16L1; Atg8; CD63; MAP1LC3B; SDCBP/syntenin-1; autophagy; chloroquine; endosome; extracellular vesicle; lysosome
  5. iScience. 2022 Mar 18. 25(3): 103877
      Malectins from the oligosaccharyltransferase (OST) complex in the endoplasmic reticulum (ER) of animal cells are involved in ER quality control and contribute to the Unfolded Protein Response (UPR). Malectins are not found in plant cells, but malectin-like domains (MLDs) are constituents of many membrane-bound receptors. In Arabidopsis thaliana, the MLD-containing receptor IOS1 promotes successful infection by filamentous plant pathogens. We show that the MLD of its exodomain retains IOS1 in the ER of plant cells and attenuates the infection-induced UPR. Expression of the MLD in the ios1-1 knockout background is sufficient to complement infection-related phenotypes of the mutant, such as increased UPR and reduced disease susceptibility. IOS1 interacts with the ER membrane-associated ribophorin HAP6 from the OST complex, and hap6 mutants show decreased pathogen-responsive UPR and increased disease susceptibility. Altogether, this study revealed a previously uncharacterized role of a plant receptor domain in the regulation of ER stress during infection.
    Keywords:  Molecular biology; Plant biology; Plant pathology
  6. Nat Commun. 2022 Mar 04. 13(1): 1181
      A large family of E3 ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade. Regulation of RING E3s depends on modulating their ability to stabilise the RING bound E2~ubiquitin conjugate in the activated (or closed) conformation. Here we report the structure of the Ark2C RING bound to both a regulatory ubiquitin molecule and an activated E2~ubiquitin conjugate. The structure shows that the RING domain and non-covalently bound ubiquitin molecule together make contacts that stabilise the activated conformation of the conjugate, revealing why ubiquitin is a key regulator of Ark2C activity. We also identify a charged loop N-terminal to the RING domain that enhances activity by interacting with both the regulatory ubiquitin and ubiquitin conjugated to the E2. In addition, the structure suggests how Lys48-linked ubiquitin chains might be assembled by Ark2C and UbcH5b. Together this study identifies features common to RING E3s, as well elements that are unique to Ark2C and related E3s, which enhance assembly of ubiquitin chains.
  7. Curr Opin Struct Biol. 2022 Feb 24. pii: S0959-440X(22)00014-8. [Epub ahead of print]73 102341
      Specificity in the ubiquitin system depends on E3 ligases, largely belonging to a handful of families discovered more than a decade ago. However, the last two years brought a quantum leap in the identification and/or mechanistic characterization of eukaryotic ubiquitin ligases, in part through implementation of activity-based chemical probes and cryo-EM. Here, we survey recent discoveries of RING-Cys-Relay, RZ-finger, and neddylated cullin-RING-ARIH RBR E3-E3 ubiquitin ligase mechanisms. These ligases transfer ubiquitin through unprecedented mechanisms-via novel catalytic domains or domain combinations-and collectively modify unconventional amino acids, non-proteinaceous bacterial lipid targets, and structurally-diverse substrates recruited to numerous cullin-RING ligases. We anticipate major expansion of the types, features, and mechanisms of E3 ligases will emerge from such chemical and structural approaches in the coming years.
  8. Plant Physiol. 2021 Dec 04. 187(4): 1916-1928
      Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues.
  9. Chembiochem. 2022 Mar 02.
      Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein posttranslational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL, for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.
    Keywords:  Fluorescent Probes; Post-translational modifications; Protein modifications; chemical protein synthesis; protein delivery
  10. ACS Cent Sci. 2022 Feb 23. 8(2): 275-281
      Ubiquitylation-the attachment of ubiquitin (Ub) to proteins in eukaryotic cells-involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics-due to a rate-limiting thioester loading step-and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates the need for Ub thioester in LACE. The chimeric E1 was subjected to directed evolution to improve its apparent second-order rate constant (k cat/K M) 400-fold. We demonstrate the utility of the chimeric E1 by site-specific transfer of mono- and oligo-Ub to various target proteins in vitro. Additionally, the chimeric E1, Ubc9, Ub, and the target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.
  11. PLoS Biol. 2022 Mar;20(3): e3001380
      Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): the guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, which are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria, and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an Δoxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2, as well as of nuclear encoded inner membrane proteins, although was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.
  12. Sci Adv. 2022 Mar 04. 8(9): eabl6293
      The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery.
  13. Cell Mol Life Sci. 2022 Mar 05. 79(3): 176
      The brain-expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntington's disease where it promotes the clearance of mutant Huntingtin. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. RTL8 also facilitates UBQLN2's nuclear translocation during heat shock. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.
    Keywords:  Nuclear protein quality control; RTL8; UBQLN2; Ubiquilin; Ubiquitin proteasome system
  14. Autophagy. 2022 Mar 01. 1-17
      TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.
    Keywords:  Autophagy; FACT; TFE3; TFEB; chaperone; histone; lysosomes
  15. J Biol Chem. 2022 Feb 26. pii: S0021-9258(22)00220-4. [Epub ahead of print] 101780
      Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum (ER) via the specific interaction of the FFAT motif with ER-resident vesicle-associated membrane protein-associated proteins (VAPs). This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins, and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3 (STARD3). Collectively, these data indicate that the creation of ISACS represents an efficient approach for studying the membrane tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.
    Keywords:  Cell biology; Confocal microscopy; Endoplasmic reticulum (ER); FFAT motif; Lipid transport; Liposome; Membrane contact sites; OSBP; STARD3; VAP
  16. J Cell Biol. 2022 May 02. pii: e202107151. [Epub ahead of print]221(5):
      Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.
  17. Cell Rep. 2022 Mar 01. pii: S2211-1247(22)00171-1. [Epub ahead of print]38(9): 110444
      Accumulation of senescent cells affects organismal aging and the prevalence of age-associated disease. Emerging evidence suggests that activation of autophagy protects against age-associated diseases and promotes longevity, but the roles and regulatory mechanisms of autophagy in cellular senescence are not well understood. Here, we identify the transcription factor, MondoA, as a regulator of cellular senescence, autophagy, and mitochondrial homeostasis. MondoA protects against cellular senescence by activating autophagy partly through the suppression of an autophagy-negative regulator, Rubicon. In addition, we identify peroxiredoxin 3 (Prdx3) as another downstream regulator of MondoA essential for mitochondrial homeostasis and autophagy. Rubicon and Prdx3 work independently to regulate senescence. Furthermore, we find that MondoA knockout mice have exacerbated senescence during ischemic acute kidney injury (AKI), and a decrease of MondoA in the nucleus is correlated with human aging and ischemic AKI. Our results suggest that decline of MondoA worsens senescence and age-associated disease.
    Keywords:  C. elegans; MondoA; Rubicon; aging; autophagy; cellular senescence; kidney; mitochondrial homeostasis; mml-1; peroxiredoxin 3
  18. Mol Cell. 2022 Feb 25. pii: S1097-2765(22)00086-7. [Epub ahead of print]
      Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.
    Keywords:  Plin2; Ubr1; amino acid; autoinhibition; hepatic steatosis; isoleucine; leucine; lipid droplet; nonalcoholic fatty liver disease; ubiquitination
  19. FEBS J. 2022 Mar 05.
      Von Willebrand Factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.
    Keywords:  GBF1; Golgi; SEC22B; SNARE; STX5; Von Willebrand Disease; Von Willebrand factor; Weibel-Palade body; endoplasmic reticulum; endothelial cell
  20. PLoS Genet. 2022 Mar 03. 18(3): e1010106
      In yeast, at least seven proteins (Ice2p, Ist2p, Scs2/22p, Tcb1-Tcb3p) affect cortical endoplasmic reticulum (ER) tethering and contact with the plasma membrane (PM). In Δ-super-tether (Δ-s-tether) cells that lack these tethers, cortical ER-PM association is all but gone. Yeast OSBP homologue (Osh) proteins are also implicated in membrane contact site (MCS) assembly, perhaps as subunits for multicomponent tethers, though their function at MCSs involves intermembrane lipid transfer. Paradoxically, when analyzed by fluorescence and electron microscopy, the elimination of the OSH gene family does not reduce cortical ER-PM association but dramatically increases it. In response to the inactivation of all Osh proteins, the yeast E-Syt (extended-synaptotagmin) homologue Tcb3p is post-transcriptionally upregulated thereby generating additional Tcb3p-dependent ER-PM MCSs for recruiting more cortical ER to the PM. Although the elimination of OSH genes and the deletion of ER-PM tether genes have divergent effects on cortical ER-PM association, both elicit the Environmental Stress Response (ESR). Through comparisons of transcriptomic profiles of cells lacking OSH genes or ER-PM tethers, changes in ESR expression are partially manifested through the induction of the HOG (high-osmolarity glycerol) PM stress pathway or the ER-specific UPR (unfolded protein response) pathway, respectively. Defects in either UPR or HOG pathways also increase ER-PM MCSs, and expression of extra "artificial ER-PM membrane staples" rescues growth of UPR mutants challenged with lethal ER stress. Transcriptome analysis of OSH and Δ-s-tether mutants also revealed dysregulation of inositol-dependent phospholipid gene expression, and the combined lethality of osh4Δ and Δ-s-tether mutations is suppressed by overexpression of the phosphatidic acid biosynthetic gene, DGK1. These findings establish that the Tcb3p tether is induced by ER and PM stresses and ER-PM MCSs augment responses to membrane stresses, which are integrated through the broader ESR pathway.
  21. Connect Tissue Res. 2022 Feb 26. 1-18
      Mutations in collagen genes cause a broad range of connective tissue pathologies. Structural mutations that impact procollagen assembly or triple helix formation and stability are a common and important mutation class. How misfolded procollagens engage with the cellular proteostasis machinery and whether they can elicit a cytotoxic unfolded protein response (UPR) is a topic of considerable research interest. Such interest is well justified since modulating the UPR could offer a new approach to treat collagenopathies for which there are no current disease mechanism-targeting therapies. This review scrutinizes the evidence underpinning the view that endoplasmic reticulum stress and chronic UPR activation contributes significantly to the pathophysiology of the collagenopathies. While there is strong evidence that the UPR contributes to the pathology for collagen X misfolding mutations, the evidence that misfolding mutations in other collagen types induce a canonical, cytotoxic UPR is incomplete. To gain a more comprehensive understanding about how the UPR amplifies to pathology, and thus what types of manipulations of the UPR might have therapeutic relevance, much more information is needed about how specific misfolding mutation types engage differentially with the UPR and downstream signaling responses. Most importantly, since the capacity of the proteostasis machinery to respond to collagen misfolding is likely to vary between cell types, reflecting their functional roles in collagen and extracellular matrix biosynthesis, detailed studies on the UPR should focus as much as possible on the actual target cells involved in the collagen pathologies.
    Keywords:  Collagen mutations; collagen misfolding; collagenopathies; endoplasmic reticulum stress; proteostasis; unfolded protein response
  22. Autophagy. 2022 Feb 28. 1-12
      Macroautophagy/autophagy is a tightly regulated catabolic process, which contributes at baseline level to cellular homeostasis, and upon its stimulation to the adaptive cellular response to intra- and extracellular stress stimuli. Decrease of autophagy activity is occurring upon aging and thought to contribute to age-related-diseases. Recently, we uncovered, upon autophagy induction, the role of de novo DNMT3A (DNA methyltransferase 3 alpha)-mediated DNA methylation on expression of the MAP1LC3 (microtubule associated protein 1 light chain 3) proteins, core components of the autophagy pathway, which resulted in reduced baseline autophagy activity. Here, we report that serine/threonine kinase ULK3 (unc-51 like kinase 3)-dependent activation of GLI1 (GLI family zinc finger 1) contributes to the transcriptional upregulation of DNMT3A gene expression upon autophagy induction, thereby bringing additional understanding of the long-term effect of autophagy induction and a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: CBZ: carbamazepine; ChIP: chromatin immunoprecipitation; Clon: clonidine; DNMT3A: DNA methyltransferase 3 alpha; GLI1: GLI family zinc finger 1; GLI2: GLI family zinc finger 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PLA: proximity ligation assay; RT-qPCR: quantitative reverse transcription PCR; shRNA: small hairpin RNA; siRNA: small interfering RNA; Treh: trehalose; ULK3: unc-51 like kinase 3.
    Keywords:  Autophagy; DNMT3A; GLI1; ULK3; transcription
  23. Oncogene. 2022 Mar 02.
      Hepatocellular carcinoma (HCC) is one of the largest causes of cancer-related deaths worldwide owing to the limitation of effective treatment options. The ubiquitin-proteasome system has been rapidly recognized as a frequent target of deregulation leading to cancers. Enhanced DNA damage response (DDR) promotes HCC growth and prevents chemosensitivity, and ubiquitin E3 ligases are key modulators in DDR. Therefore, a better understanding of how E3 ligases regulate cell growth and DNA damage may provide novel insights in understanding the oncogenic mechanism and improving the efficacy of DNA damage therapeutic agents. Here, we performed a high-content RNAi screening targeting 52 DDR-related E3 ligases in HCC and found that ring finger protein 4 (RNF4) was essential for HCC growth. RNF4 was highly expressed in HCC tissues, and the expression levels of RNF4 were associated with poor outcomes. RNF4 silencing significantly suppressed the cell growth, and subsequently induced G2/M arrest and apoptosis of HCC cells in vitro; RNF4 silencing also demonstrated the tumor-suppressive efficacy on HCC in vivo. Moreover, RNF4 silencing increased DNA damage, and rendered HCC cells more sensitive to DNA damage drugs and radiation. We found RNF4 functionally interacts with p62, and mechanistic analyses indicated that RNF4 silencing triggered the nuclear enrichment of p62. Moreover, the p62 nuclear targeting was required for increased DNA damage and growth suppression mediated by RNF4 silencing. Thus, our findings suggest RNF4 is essential for HCC proliferation via preventing nuclear translocation of p62. RNF4 silencing promotes DNA damage and may serve as a novel strategy to suppress cell growth and increase the sensitivity of DNA damage therapeutic agents in HCC.
  24. Cell Chem Biol. 2022 Feb 28. pii: S2451-9456(22)00086-1. [Epub ahead of print]
      The spatial arrangement of newly synthesized transcriptome in eukaryotic cells underlies various biological processes including cell proliferation and differentiation. In this study, we combine metabolic incorporation of electron-rich ribonucleosides (e.g., 6-thioguanosine and 4-thiouridine) with a peroxidase-mediated proximity-dependent RNA labeling technique (APEX-seq) to develop a sensitive method, termed MERR APEX-seq, for selectively profiling newly transcribed RNAs at specific subcellular locations in live cells. We demonstrate that MERR APEX-seq is 20-fold more efficient than APEX-seq and offers both high spatial specificity and high coverage in mitochondrial matrix. At the ER membrane, 91% of the transcripts captured by MERR APEX-seq encode for secretory pathway proteins, thus demonstrating the high spatial specificity of MERR APEX-seq in open subcellular compartments. Application of MERR APEX-seq to the nuclear lamina of human cells reveals a local transcriptome of 1,012 RNAs, many of which encode for nuclear proteins involved in histone modification, chromosomal structure maintenance, and RNA processing.
    Keywords:  APEX-seq; RNA labeling; nascent transcriptome; nuclear lamina
  25. Cell Rep Med. 2022 Feb 15. 3(2): 100502
      Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.
    Keywords:  HSP90; combination therapy; glycolysis; metabolism; prostate cancer; therapeutics; therapy
  26. Autophagy. 2022 Feb 28. 1-2
      Macroautophagy/autophagy-related protein Atg8/LC3 is important for autophagosome biogenesis and required for selective degradation of various substrates. In our recent study, we performed a yeast two-hybrid screening to identify proteins that interact with Atg8a, the Drosophila homolog of Atg8/LC3. The screening identified several Atg8a-interacting proteins. These proteins include: i) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, ref(2)P and key (Kenny); ii) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4; and iii) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1. We showed that Tak1, as well as its co-activator, Tab2, both interact with Atg8a and are substrates for selective autophagic clearance. We also determined that SH3PX1 interacts with Tab2 and is necessary for the effective regulation of the immune-deficiency (IMD) pathway. Our findings suggest a mechanism for the regulatory interactions between Tak1-Tab2-SH3PX1 and Atg8a, which contribute to the fine-tuning of the IMD pathway.
    Keywords:  Autophagy; IMD; LIR-motif; Sh3px1; Tab2; Tak1; inflammation
  27. ACS Omega. 2022 Feb 22. 7(7): 5615-5624
      Ubiquitination is a modification of proteins that has a powerful impact on protein function along with other cellular functions. This reaction is regulated through major enzymes, including E3 ligase as a chief enzyme. The Cullin-5 ubiquitin ligase (Cul5) possesses a variety of substrates that maintain the process of ubiquitination as well as proteasomal degradation. It regulates cell development, proliferation, and other physiological tasks in the human body. Moreover, it has been discovered that the expression of Cul5 plays a significant role in specific cancer cells while affecting the progression of tumor cells. This review is based on current knowledge about Cul5 and its expression, signaling pathways, regulation, virus-related responses, and inhibitors for therapeutic strategies.
  28. Cell Death Dis. 2022 Mar 02. 13(3): 197
      Multiple myeloma (MM) remains an incurable plasma cell cancer characterized by abnormal secretion of monoclonal immunoglobulins. The molecular mechanism that regulates the drug sensitivity of MM cells is being intensively studied. Here, we report an unexpected finding that the protein encoded by neural precursor cell-expressed developmentally downregulated gene 4L (NEDD4L), which is a HECT E3 ligase, binds the 19S proteasome, limiting its proteolytic function and enhancing autophagy. Suppression of NEDD4L expression reduced bortezomib (Bor) sensitivity in vitro and in vivo, mainly through autophagy inhibition mediated by low NEDD4L expression, which was rescued by an autophagy activator. Clinically, elevated expression of NEDD4L is associated with a considerably increased probability of responding to Bor, a prolonged response duration, and improved overall prognosis, supporting both the use of NEDD4L as a biomarker to identify patients most likely to benefit from Bor and the regulation of NEDD4L as a new approach in myeloma therapy.
  29. Nat Immunol. 2022 Feb 28.
      Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.
  30. Curr Opin Struct Biol. 2022 Feb 23. pii: S0959-440X(22)00013-6. [Epub ahead of print]73 102340
      Proteins encounter frequent molecular interactions in biological environments. Computer simulations have become an increasingly important tool in providing mechanistic insights into how such interactions in vivo relate to their biological function. The review here focuses on simulations describing protein assembly and molecular crowding effects as two important aspects that are distinguished mainly by how specific and long-lived protein contacts are. On the topic of crowding, recent simulations have provided new insights into how crowding affects protein folding and stability, modulates enzyme activity, and affects diffusive properties. Recent studies of assembly processes focus on assembly pathways, especially for virus capsids, amyloid aggregation pathways, and the role of multivalent interactions leading to phase separation. Also, discussed are technical challenges in achieving increasingly realistic simulations of interactions in cellular environments.