bims-proteo Biomed News
on Proteostasis
Issue of 2020‒07‒12
forty-seven papers selected by
Eric Chevet

  1. Anal Biochem. 2020 Jul 01. pii: S0003-2697(20)30361-4. [Epub ahead of print] 113829
    Estabrooks SK, Brodsky JL.
      Soluble secreted proteins and membrane proteins are subjected to protein quality control pathways during their synthesis in the endoplasmic reticulum (ER) and delivery to other destinations. Foremost among these quality control pathways is the selection of misfolded proteins for ER-associated degradation (ERAD). A growing number of diseases, including Cystic Fibrosis, are linked to the ERAD pathway. In most cases, a membrane protein known as the Cystic Fibrosis Transmembrane Conductance Regulator, or CFTR, is prematurely degraded by ERAD. Cell-based assays and in vitro studies have elucidated factors required for the recognition and degradation of CFTR, yet mechanistic details on how these factors target specific disease-causing variants is limited. Given the possibility that variants might exhibit unique susceptibilities to ubiquitin modification, which is required for proteasome-mediated degradation, we devised an assay that recapitulates this event. Here, we demonstrate that ER-enriched membranes from transfected human cells support CFTR ubiquitination when combined with radiolabeled ubiquitin and isolated enzymes in the ubiquitination cascade. We also show that select disease-causing variants are ubiquitinated more extensively than wild-type channels and to varying degrees. Our system provides a platform to examine how other purified factors impact CFTR ubiquitination and the ubiquitination of additional disease-associated membrane proteins.
    Keywords:  ERAD; F508del CFTR; cystic fibrosis; protein quality control; ubiquitination
  2. Data Brief. 2020 Aug;31 105931
    Kriegler T, Lang S, Notari L, Hessa T.
      The Prion protein (PrP) is a highly conserved cell surface glycoprotein. To enter the secretory pathway, the PrP precursor relies on the Sec61 complex and multiple accessory factors all gathering at the membrane of the Endoplasmic reticulum (ER). PrP topogenesis results in the formation of different PrP isoforms. Aside from the typical secretory variant (SecPrP) different pathognomonic, membrane-embedded variants (NtmPrP and CtmPrP) that are associated with neurodegenerative diseases can be found [1]. In this article, we provide supportive data related to "Prion Protein Translocation Mechanism Revealed by Pulling Force Studies" (Kriegler et al., May 2020)[2], where we utilize Xbp1 arrest peptide (AP)-mediated ribosomal stalling to study the co-translational folding experienced by PrP during its insertion into the ER. We measure translocation efficiency and characterize the force exerted on PrP nascent chain so called "pulling force profile". Here, we describe the method of AP-mediated ribosomal stalling assay together with additional experimental data to the main article. Furthermore, we describe the combination of AP-mediated ribosomal stalling and semi-permeabilized Hela cells (SPCs) as ER membrane source. Using this experimental set-up one can directly determine the contribution of a specific membrane component, e.g. subunits of the ER protein translocase, as pulling factor exerting force on the PrP nascent chain. The data presented here covers (a) the SDS-PAGE gel images visualized by autoradiography, (b) quantification of the different populations of PrP species observed in the AP-mediated ribosomal stalling method, and (c) calculation formulas of the pulling force profiles measured in SPCs in comparison to dog pancreas microsomes as ER membrane donor. Finally, Western Blot analysis and quantification of siRNA knockdown levels compared to control conditions of various translocation components are shown.
    Keywords:  Cotranslational folding; Prion protein; Pulling force; XBP1-arrest peptide; semi-permeabilized cells
  3. Int J Mol Sci. 2020 Jul 04. pii: E4757. [Epub ahead of print]21(13):
    Pellegrini P, Selvaraju K, Faustini E, Mofers A, Zhang X, Ternerot J, Schubert A, Linder S, D Arcy P.
      The proteasome is a validated target of cancer therapeutics. Inhibition of proteasome activity results in the activation of the unfolded protein response (UPR) characterized by phosphorylation of eukaryotic initiation factor 2α (eIF2α), global translational arrest, and increased expression of the proapoptotic CHOP (C/EBP homologous protein) protein. Defects in the UPR response has been reported to result in altered sensitivity of tumor cells to proteasome inhibitors. Here, we characterized the effects of the deubiquitinase (DUB) inhibitor VLX1570 on protein homeostasis, both at the level of the UPR and on protein translation, in acute lymphoblastic leukemia (ALL). Similar to the 20S inhibitor bortezomib, VLX1570 induced accumulation of polyubiquitinated proteins and increased expression of the chaperone Grp78/Bip in ALL cells. Both compounds induced cleavage of PARP (Poly (ADP-ribose) polymerase) in ALL cells, consistent with induction of apoptosis. However, and in contrast to bortezomib, VLX1570 treatment resulted in limited induction of the proapoptotic CHOP protein. Translational inhibition was observed by both bortezomib and VLX1570. We report that in distinction to bortezomib, suppression of translation by VXL1570 occurred at the level of elongation. Increased levels of Hsc70/Hsp70 proteins were observed on polysomes following exposure to VLX1570, possibly suggesting defects in nascent protein folding. Our findings demonstrate apoptosis induction in ALL cells that appears to be uncoupled from CHOP induction, and show that VLX1570 suppresses protein translation by a mechanism distinct from that of bortezomib.
    Keywords:  acute lymphocytic leukemia; bortezomib; proteasome; translation
  4. Acta Neuropathol. 2020 Jul 08.
    García-Huerta P, Troncoso-Escudero P, Wu D, Thiruvalluvan A, Cisternas-Olmedo M, Henríquez DR, Plate L, Chana-Cuevas P, Saquel C, Thielen P, Longo KA, Geddes BJ, Lederkremer GZ, Sharma N, Shenkman M, Naphade S, Sardi SP, Spichiger C, Richter HG, Court FA, Tshilenge KT, Ellerby LM, Wiseman RL, Gonzalez-Billault C, Bergink S, Vidal RL, Hetz C.
      Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.
  5. Mol Cell. 2020 Jun 30. pii: S1097-2765(20)30425-1. [Epub ahead of print]
    Hu X, Wang L, Wang Y, Ji J, Li J, Wang Z, Li C, Zhang Y, Zhang ZR.
      Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.
    Keywords:  RNF126; VCP; endoplasmic reticulum-associated degradation; membrane protein; p97/valosin-containing protein; proteasome; ubiquitination
  6. Elife. 2020 Jul 10. pii: e56584. [Epub ahead of print]9
    Xu L, Wang X, Zhou J, Qiu Y, Shang W, Liu JP, Wang L, Tong C.
      Endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, mitochondrial dynamics, and autophagosome biogenesis. However, the molecular organization, functions, and regulation of ERMCS are not fully understood in higher eukaryotes. Also, the physiological roles of altered ERMCSs are not well defined. In this study, we found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.
    Keywords:  D. melanogaster; cell biology; human
  7. Sci Adv. 2020 Jun;6(26): eaaz9805
    Higuchi-Sanabria R, Shen K, Kelet N, Frankino PA, Durieux J, Bar-Ziv R, Sing CN, Garcia EJ, Homentcovschi S, Sanchez M, Wu R, Tronnes SU, Joe L, Webster B, Ahilon-Jeronimo A, Monshietehadi S, Dallarda S, Pender C, Pon LA, Zoncu R, Dillin A.
      Recent work has highlighted the fact that lysosomes are a critical signaling hub of metabolic processes, providing fundamental building blocks crucial for anabolic functions. How lysosomal functions affect other cellular compartments is not fully understood. Here, we find that lysosomal recycling of the amino acids lysine and arginine is essential for proper ER quality control through the UPRER. Specifically, loss of the lysine and arginine amino acid transporter LAAT-1 results in increased sensitivity to proteotoxic stress in the ER and decreased animal physiology. We find that these LAAT-1-dependent effects are linked to glycine metabolism and transport and that the loss of function of the glycine transporter SKAT-1 also increases sensitivity to ER stress. Direct lysine and arginine supplementation, or glycine supplementation alone, can ameliorate increased ER stress sensitivity found in laat-1 mutants. These data implicate a crucial role in recycling lysine, arginine, and glycine in communication between the lysosome and ER.
  8. Cell Stress Chaperones. 2020 Jul 06.
    Moghadami AA, Aboutalebi Vand Beilankouhi E, Kalantary-Charvadeh A, Hamzavi M, Mosayyebi B, Sedghi H, Ghorbani Haghjo A, Nazari Soltan Ahmad S.
      Non-small cell lung cancer is the most common type of lung cancer, accounting for more than 80% of this tumor. Ubiquitin-specific protease (USP) 14 is one of the 100 deubiquitinating enzymes that is overexpressed in lung cancer and has been validated as a therapeutic target. The aim of this study is to determine whether the accumulation of ubiquitinated proteins results in endoplasmic reticulum (ER) stress-mediated autophagy. To inhibit USP-14, A549 lung cancer cells were treated with USP-14 siRNA and IU1-47 (20 μM). The protein level, mRNA expression, and cell cycle analysis were evaluated using Western blot, real-time PCR, and flow cytometry, respectively. We found that treating A549 cells with USP14 inhibitors significantly reduced the proliferation rate and induced cell cycle arrest at G2/M phase. We also found that USP14 inhibitors did not induce apoptosis but actually induced autophagy through accumulation of ubiquitinated proteins/ER stress/unfolded protein response (UPR) axis. Moreover, we have for the first time demonstrated that the USP14 inhibition induces ER stress-mediated autophagy in A549 cells by activation of c-Jun N-terminal kinase 1 (JNK1). In conclusion, the current investigation represents a new mechanism by which inhibition of USP14 triggers autophagy via ER stress-mediated UPR in A549 cells.
    Keywords:  Autophagy; Endoplasmic reticulum stress; JNK; NSCLC; Ubiquitin-proteasome
  9. J Mol Med (Berl). 2020 Jul 06.
    Zou J, Jones RJ, Wang H, Kuiatse I, Shirazi F, Manasanch EE, Lee HC, Sullivan R, Fung L, Richard N, Erdman P, Torres E, Hecht D, Lam I, McElwee B, Chourasia AH, Chan KWH, Mercurio F, Stirling DI, Orlowski RZ.
      Small molecules targeting the cereblon-containing E3 ubiquitin ligase including thalidomide, lenalidomide, and pomalidomide modulate turnover of downstream client proteins and demonstrate pre-clinical and clinical anti-myeloma activity. Different drugs that engage with cereblon hold the potential of unique phenotypic effects, and we therefore studied the novel protein homeostatic modulator (PHM™) BTX306 with a unique thiophene-fused scaffold bearing a substituted phenylurea and glutarimide. This agent much more potently reduced human-derived myeloma cell line viability, with median inhibitory concentrations in the single nanomolar range versus micromolar values for lenalidomide or pomalidomide, and more potently activated caspases 3/8/9. While lenalidomide and pomalidomide induced greater degradation of Ikaros and Aiolos in myeloma cells, BTX306 more potently reduced levels of GSPT1, eRF1, CK1α, MCL-1, and c-MYC. Suppression of cereblon or overexpression of Aiolos or Ikaros induced relative resistance to BTX306, and this agent did not impact viability of murine hematopoietic cells in an in vivo model, demonstrating its specificity for human cereblon. Interestingly, BTX306 did show some reduced activity in lenalidomide-resistant cell line models but nonetheless retained its nanomolar potency in vitro, overcame bortezomib resistance, and was equipotent against otherwise isogenic cell line models with either wild-type or knockout TP53. Finally, BTX306 demonstrated strong activity against primary CD138-positive plasma cells, showed enhanced anti-proliferative activity in combination with bortezomib and dexamethasone, and was effective in an in vivo systemic model of multiple myeloma. Taken together, the data support further translational studies of BTX306 and its derivatives to the clinic for patients with relapsed and/or refractory myeloma. KEY MESSAGES: BTX306 has a unique thiophene-fused scaffold bearing phenylurea and glutarimide. BTX306 is more potent against myeloma cells than lenalidomide or pomalidomide. BTX306 overcomes myeloma cell resistance to lenalidomide or bortezomib in vitro. BTX306 is active against primary myeloma cells, and shows efficacy in vivo.
    Keywords:  Aiolos; Cereblon; GSPT1; Ikaros; Multiple myeloma; Protein homeostatic modulator
  10. mBio. 2020 Jul 07. pii: e00915-20. [Epub ahead of print]11(4):
    Augusto L, Martynowicz J, Amin PH, Alakhras NS, Kaplan MH, Wek RC, Sullivan WJ.
      Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here, we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further identify a novel role for the host ER stress sensor protein IRE1 in Toxoplasma pathogenesis. Upon infection, Toxoplasma activates IRE1, engaging its noncanonical role in actin remodeling through the binding of filamin A. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo Our study has identified novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis.IMPORTANCE Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this report, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration involves not the enzymatic activity of IRE1 but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.
    Keywords:  IRE1; PERK; Toxoplasma ; UPR; cell migration; filamin A; host-pathogen interactions; parasites; pathogenesis
  11. Life Sci. 2020 Jul 02. pii: S0024-3205(20)30791-8. [Epub ahead of print] 118041
    Thacker G, Mishra M, Sharma A, Singh AK, Sanyal S, Trivedi AK.
      AIM: Transcription factor CCAAT/Enhancer binding protein alpha (C/EBPα) is a key regulator of myeloid differentiation, granulopoiesis in particular. Although CEBPA mutations are found in more than 10% in AML, functional inhibition of C/EBPα protein is also widely observed in AML. Here, we sought to examine if SKP2, an aberrantly enhanced E3 ubiquitin ligase in primary AMLs inhibits C/EBPα stability to induce differentiation block.MAIN METHODS: Here we employed cell based assays such transfections, immunoblotting, co-immunoprecipitation, luciferase and gel shift assays along with differentiation assays to investigate SKP2 regulated C/EBPα protein stability in acute myeloid leukemia.
    KEY FINDINGS: Here we discovered that oncogenic E3 ubiquitin ligase SCFskp2 ubiquitinates and destabilizes C/EBPα in a proteasome-dependent manner. Our data demonstrates that SKP2 physically interacts with C-terminal of C/EBPα and promotes its K48-linked ubiquitination-mediated degradation leading to its reduced transactivation potential, DNA binding ability and cellular functions. We further show that while overexpression of SKP2 inhibits both ectopic as well as endogenous C/EBPα in heterologous (HEK293T) as well as myeloid leukemia cells respectively, SKP2 depletion restores endogenous C/EBPα leading to reduced colony formation and enhanced myeloid differentiation of myeloid leukemia cells. Using Estradiol-inducible K562-C/EBPα-ER cells as yet another model of granulocytic differentiation, we further confirmed that SKP2 overexpression indeed inhibits granulocytic differentiation by mitigating C/EBPα stability.
    SIGNIFICANCE: Our findings identify SKP2 as a potential negative regulator of C/EBPα stability and function in AML which suggests that SKP2 can be potentially targeted in AML to restore C/EBPα and overcome differentiation block.
    Keywords:  Acute myeloid leukemia; C/EBPα; Myeloid differentiation; SKP2; Ubiquitination
  12. Crit Rev Biochem Mol Biol. 2020 Jul 07. 1-32
    Qin X, Denton WD, Huiting LN, Smith KS, Feng H.
      During malignant transformation and cancer progression, tumor cells face both intrinsic and extrinsic stress, endoplasmic reticulum (ER) stress in particular. To survive and proliferate, tumor cells use multiple stress response pathways to mitigate ER stress, promoting disease aggression and treatment resistance. Among the stress response pathways is ER-associated degradation (ERAD), which consists of multiple components and steps working together to ensure protein quality and quantity. In addition to its established role in stress responses and tumor cell survival, ERAD has recently been shown to regulate tumor immunity. Here we summarize current knowledge on how ERAD promotes protein degradation, regulates immune cell development and function, participates in antigen presentation, exerts paradoxical roles on tumorigenesis and immunity, and thus impacts current cancer therapy. Collectively, ERAD is a critical protein homeostasis pathway intertwined with cancer development and tumor immunity. Of particular importance is the need to further unveil ERAD's enigmatic roles in tumor immunity to develop effective targeted and combination therapy for successful treatment of cancer.
    Keywords:  Cancer; ER stress; ERAD; immune cells; tumor immunity
  13. PLoS One. 2020 ;15(7): e0235864
    Bauer A, Santen L, Schmitt MJ, Shaebani MR, Becker B.
      In eukaryotic cells, KDEL receptors (KDELRs) facilitate the retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi compartment back to the ER. Apart from the well-documented retention function, recent findings reveal that the cellular KDELRs have more complex roles, e.g. in cell signalling, protein secretion, cell adhesion and tumorigenesis. Furthermore, several studies suggest that a sub-population of KDELRs is located at the cell surface, where they could form and internalize KDELR/cargo clusters after K/HDEL-ligand binding. However, so far it has been unclear whether there are species- or cell-type-specific differences in KDELR clustering. By comparing ligand-induced KDELR clustering in different mouse and human cell lines via live cell imaging, we show that macrophage cell lines from both species do not develop any clusters. Using RT-qPCR experiments and numerical analysis, we address the role of KDELR expression as well as endocytosis and exocytosis rates on the receptor clustering at the plasma membrane and discuss how the efficiency of directed transport to preferred docking sites on the membrane influences the exponent of the power-law distribution of the cluster size.
  14. Cell Mol Life Sci. 2020 Jul 10.
    Montenegro-Venegas C, Fienko S, Anni D, Pina-Fernández E, Frischknecht R, Fejtova A.
      Proteasomes are protein complexes that mediate controlled degradation of damaged or unneeded cellular proteins. In neurons, proteasome regulates synaptic function and its dysfunction has been linked to neurodegeneration and neuronal cell death. However, endogenous mechanisms controlling proteasomal activity are insufficiently understood. Here, we describe a novel interaction between presynaptic scaffolding protein bassoon and PSMB4, a β subunit of the 20S core proteasome. Expression of bassoon fragments that interact with PSMB4 in cell lines or in primary neurons attenuates all endopeptidase activities of cellular proteasome and induces accumulation of several classes of ubiquitinated and non-ubiquitinated substrates of the proteasome. Importantly, these effects are distinct from the previously reported impact of bassoon on ubiquitination and autophagy and might rely on a steric interference with the assembly of the 20S proteasome core. In line with a negative regulatory role of bassoon on endogenous proteasome we found increased proteasomal activity in the synaptic fractions prepared from brains of bassoon knock-out mice. Finally, increased activity of proteasome and lower expression levels of synaptic substrates of proteasome could be largely normalized upon expression of PSMB4-interacting fragments of bassoon is the name of the protein bassoon in neurons derived from bassoon deficient mice. Collectively, we propose that bassoon interacts directly with proteasome to control its activity at presynapse and thereby it contributes to a compartment-specific regulation of neuronal protein homeostasis. These findings provide a mechanistic explanation for the recently described link of bassoon is the name of the protein bassoon to human diseases associated with pathological protein aggregation. Presynaptic cytomatrix protein bassoon (Bsn) interacts with PSMB4, the β7 subunit of 20S core proteasome, via three independent interaction interfaces. Bsn inhibits proteasomal proteolytic activity and degradation of different classes of proteasomal substrates presumably due to steric interference with the assembly of 20S core of proteasome. Upon Bsn deletion in neurons, presynaptic substrates of the proteasome are depleted, which can be reversed upon expression of PSMB4-interacting interfaces of Bsn. Taken together, bsn controls the degree of proteasome degradation within the presynaptic compartment and thus, contributes to the regulation of synaptic proteome.
    Keywords:  Cytomatrix at the active zone; Protein degradation; Proteostasis; Synapse; Ubiquitin–proteasome system
  15. Front Cell Dev Biol. 2020 ;8 506
    Thevenon D, Seffouh I, Pillet C, Crespo-Yanez X, Fauvarque MO, Taillebourg E.
      The c-Myc oncogene is a transcription factor that regulates the expression of a very large set of genes mainly involved in cell growth and proliferation. It is overexpressed in more than 70% of human cancers, illustrating the importance of keeping its levels and activity under control. The ubiquitin proteasome system is a major regulator of MYC levels in humans as well as in model organisms such as Drosophila melanogaster. Although the E3 ligases that promote MYC ubiquitination have been largely investigated, the identity and the role of the deubiquitinating enzymes, which counteract their action is only beginning to be unraveled. Using isoform-specific CRISPR-Cas9 mutagenesis, we show that the Drosophila homolog of the Ubiquitin Specific Protease USP36 has different isoforms with specific sub-cellular localizations and that the nucleolar dUSP36-D isoform is specifically required for cell and organismal growth. We also demonstrate that this isoform interacts with dMYC and the E3 ligase AGO and regulates their stability and ubiquitination levels. Furthermore, we show that dUSP36 is ubiquitinated by AGO and is able to self-deubiquitinate. Finally, we provide in vivo evidence supporting the functional relevance of these regulatory relationships. Together these results reveal that dMYC, AGO and dUSP36 form a tripartite, evolutionary conserved complex that acts as a regulatory node to control dMYC protein levels.
    Keywords:  CRISPR/Cas9; MYC stability; cell growth; deubiquitinase (DUB); ubiquitin (Ub)
  16. Proc Natl Acad Sci U S A. 2020 Jul 09. pii: 202006238. [Epub ahead of print]
    Peters CH, Myers ME, Juchno J, Haimbaugh C, Bichraoui H, Du Y, Bankston JR, Walker LA, Proenza C.
      Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.
    Keywords:  HCN channel; IRAG; LRMP; ion channel; sinoatrial node
  17. Front Cell Dev Biol. 2020 ;8 428
    Xu L, Wang X, Tong C.
      Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are dynamic contact regions with a distance of 10-30 nm between the endoplasmic reticulum and mitochondria. Endoplasmic reticulum-mitochondria contact sites regulate various biological processes, including lipid transfer, calcium homeostasis, autophagy, and mitochondrial dynamics. The dysfunction of ERMCS is closely associated with various neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. In this review, we will summarize the current knowledge of the components and organization of ERMCSs, the methods for monitoring ERMCSs, and the physiological functions of ERMCSs in different model systems. Additionally, we will emphasize the current understanding of the malfunction of ERMCSs and their potential roles in neurodegenerative diseases.
    Keywords:  autophagy; contact sites; endoplasmic reticulum; mitochdonrion; neurodegeneration
  18. Int J Mol Sci. 2020 Jul 08. pii: E4844. [Epub ahead of print]21(14):
    Degrugillier F, Aissat A, Prulière-Escabasse V, Bizard L, Simonneau B, Decrouy X, Jiang C, Rotin D, Fanen P, Simon S.
      Cystic Fibrosis is a lethal monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The most frequent mutation is the deletion of phenylalanine at position 508 of the protein. This F508del-CFTR mutation leads to misfolded protein that is detected by the quality control machinery within the endoplasmic reticulum and targeted for destruction by the proteasome. Modulating quality control proteins as molecular chaperones is a promising strategy for attenuating the degradation and stabilizing the mutant CFTR at the plasma membrane. Among the molecular chaperones, the small heat shock protein HspB1 and HspB4 were shown to promote degradation of F508del-CFTR. Here, we investigated the impact of HspB5 expression and phosphorylation on transport to the plasma membrane, function and stability of F508del-CFTR. We show that a phosphomimetic form of HspB5 increases the transport to the plasma membrane, function and stability of F508del-CFTR. These activities are further enhanced in presence of therapeutic drugs currently used for the treatment of cystic fibrosis (VX-770/Ivacaftor, VX-770+VX-809/Orkambi). Overall, this study highlights the beneficial effects of a phosphorylated form of HspB5 on F508del-CFTR rescue and its therapeutic potential in cystic fibrosis.
    Keywords:  CFTR; CRYAB; HspB5; alpha B-crystallin; cystic fibrosis; phosphorylation
  19. Biochemistry. 2020 Jul 06.
    Pellowe GA, Findlay HE, Lee K, Gemeinhardt TM, Blackholly LR, Reading E, Booth PJ.
      Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snap shots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid (DIBMA) co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milleu.
  20. Methods Mol Biol. 2020 ;2177 23-33
    Stefano G, Brandizzi F.
      The endoplasmic reticulum (ER) is one of the most abundant endomembrane compartments and is in close association with most of the other organelles. In mammalian and yeast cells, the physiological roles and the molecular machineries underlying such association have only recently begun to emerge. In plant cells, recent live-cell confocal imaging and electron microscopy studies have established that endosomes are associated with the ER [1]. Here, we describe confocal imaging methods and software to analyze ER-endosome association in plant cells.
    Keywords:  Endoplasmic reticulum; FRET; contact sites; correlation analyses; endosomes
  21. Trends Biochem Sci. 2020 Jul 02. pii: S0968-0004(20)30151-1. [Epub ahead of print]
    Zhao Q, Gao SM, Wang MC.
      Lysosomes transcend the role of degradation stations, acting as key nodes for interorganelle crosstalk and signal transduction. Lysosomes communicate with the nucleus through physical proximity and functional interaction. In response to external and internal stimuli, lysosomes actively adjust their distribution between peripheral and perinuclear regions and modulate lysosome-nucleus signaling pathways; in turn, the nucleus fine-tunes lysosomal biogenesis and functions through transcriptional controls. Changes in coordination between these two essential organelles are associated with metabolic disorders, neurodegenerative diseases, and aging. In this review, we address recent advances in lysosome-nucleus communication by multi-tiered regulatory mechanisms and discuss how these regulations couple metabolic inputs with organellar motility, cellular signaling, and transcriptional network.
    Keywords:  lysosomal adaptation; lysosomal metabolites; lysosome positioning; lysosome-to-nucleus signaling; transcription factors
  22. Cells. 2020 Jul 07. pii: E1632. [Epub ahead of print]9(7):
    Bommer UA, Telerman A.
      Translationally controlled tumor protein (TCTP), also called histamine releasing factor (HRF) or fortilin, is a multifunctional protein present in almost all eukaryotic organisms. TCTP is involved in a range of basic cell biological processes, such as promotion of growth and development, or cellular defense in response to biological stresses. Cellular TCTP levels are highly regulated in response to a variety of physiological signals, and regulatory mechanism at various levels have been elucidated. Given the importance of TCTP in maintaining cellular homeostasis, it is not surprising that dysregulation of this protein is associated with a range of disease processes. Here, we review recent progress that has been made in the characterisation of the basic biological functions of TCTP, in the description of mechanisms involved in regulating its cellular levels and in the understanding of dysregulation of TCTP, as it occurs in disease processes such as cancer.
    Keywords:  TCTP (HRF, fortilin); autophagy; biological stress reactions; cancer; cardiovascular diseases; growth and development; regulated protein degradation; regulation of protein synthesis
  23. J Biol Chem. 2020 Jul 09. pii: jbc.AC120.014940. [Epub ahead of print]
    Marinko JT, Carter BD, Sanders CR.
      Charcot-Marie-Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ~1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third wild-type PMP22 allele. In rodent models overexpressing the peripheral myelin protein 22 (PMP22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum (ER) quality control, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of wild-type or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells as well as PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mis-trafficking of PMP22, which is likely a contributor to disease pathology and progression.
    Keywords:  Charcot-Marie-Tooth disease (CMT); cell surface protein; flow cytometry; membrane protein; membrane trafficking; myelin; peripheral neuropathy; protein folding; protein misfolding
  24. Int J Mol Sci. 2020 Jun 30. pii: E4654. [Epub ahead of print]21(13):
    Linders PTA, Peters E, Ter Beest M, Lefeber DJ, van den Bogaart G.
      Glycosylation is an important post-translational modification for both intracellular and secreted proteins. For glycosylation to occur, cargo must be transported after synthesis through the different compartments of the Golgi apparatus where distinct monosaccharides are sequentially bound and trimmed, resulting in increasingly complex branched glycan structures. Of utmost importance for this process is the intraorganellar environment of the Golgi. Each Golgi compartment has a distinct pH, which is maintained by the vacuolar H+-ATPase (V-ATPase). Moreover, tethering factors such as Golgins and the conserved oligomeric Golgi (COG) complex, in concert with coatomer (COPI) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion, efficiently deliver glycosylation enzymes to the right Golgi compartment. Together, these factors maintain intra-Golgi trafficking of proteins involved in glycosylation and thereby enable proper glycosylation. However, pathogenic mutations in these factors can cause defective glycosylation and lead to diseases with a wide variety of symptoms such as liver dysfunction and skin and bone disorders. Collectively, this group of disorders is known as congenital disorders of glycosylation (CDG). Recent technological advances have enabled the robust identification of novel CDGs related to membrane trafficking components. In this review, we highlight differences and similarities between membrane trafficking-related CDGs.
    Keywords:  Golgi apparatus; congenital disorders of glycosylation; glycosylation; membrane trafficking; post-translational modification; secretory pathway
  25. FEBS J. 2020 Jul 05.
    Iwai K.
      Ubiquitination is a reversible post-translational modification that regulates function of conjugated proteins by decorating with ubiquitin chains -polymer of ubiquitin- in most cases. The discovery of linear ubiquitin chains and the linear ubiquitin chain assembly complex (LUBAC) ubiquitin ligase complex can be considered as paradigm shift in the ubiquitin research because the linear ubiquitin chain is generated via the N-terminal Met of ubiquitin, although the other ubiquitin chains are generated via one of seven Lys residues in ubiquitin. Moreover, ubiquitination is distributed throughout eukaryotic kingdoms, however, no linear ubiquitination could be found in lower eukaryotes including yeasts. Although involvement of ubiquitination in proteolysis is well-documented, linear ubiquitination plays crucial roles in immune signaling and cell death regulation. Moreover, dysregulation of LUBAC-mediated linear ubiquitination underlies various human diseases including autoinflammation and cancer. Here, I introduce how linear ubiquitination was discovered and outline a brief history of linear ubiquitination research.
    Keywords:  LUBAC; NF-κB; cancer; cell death; immunodeficiency; infectious diseases; linear ubiquitination
  26. Life Sci Alliance. 2020 Aug;pii: e202000768. [Epub ahead of print]3(8):
    Rusilowicz-Jones EV, Jardine J, Kallinos A, Pinto-Fernandez A, Guenther F, Giurrandino M, Barone FG, McCarron K, Burke CJ, Murad A, Martinez A, Marcassa E, Gersch M, Buckmelter AJ, Kayser-Bricker KJ, Lamoliatte F, Gajbhiye A, Davis S, Scott HC, Murphy E, England K, Mortiboys H, Komander D, Trost M, Kessler BM, Ioannidis S, Ahlijanian MK, Urbé S, Clague MJ.
      The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.
  27. J Cell Biochem. 2020 Jul 06.
    Su L, Zhao T, Li H, Li H, Su X, Ba X, Zhang Y, Huang B, Lu J, Li X.
      5O-GlcNAc transferase (OGT) is the enzyme catalyzing protein O-GlcNAcylation by addition of a single O-linked-β-N-acetylglucosamine molecule (O-GlcNAc) to nuclear and cytoplasmic targets, and it uses uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor. As UDP-GlcNAc is the final product of the nutrient-sensing hexosamine signaling pathway, overexpression or knockout of ogt in mammals or invertebrate models influences cellular nutrient-response signals and increases susceptibility to chronic diseases of aging. Evidence shows that OGT expression levels decrease in tissues of older mice and rats. However, how OGT expression is modulated in the aging process remains poorly understood. In Caenorhabditis elegans, the exclusive mammalian OGT ortholog OGT-1 is crucial for lifespan control. Here, we observe that worm OGT-1 expression gradually reduces during aging. By combining prediction via the "MATCH" algorithm and luciferase reporter assays, GATA factor ELT-2, the homolog of human GATA4, is identified as a transcriptional factor driving OGT-1 expression. Chromatin immunoprecipitation-quantitative polymerase chain reaction and electrophoretic mobility shift assays show ELT-2 directly binds to and activates the ogt-1 promoter. Knockdown of elt-2 decreases the global O-GlcNAc modification level and reduces the lifespan of wild-type worms. The reduction in lifespan caused by elt-2 RNA interference is abrogated by the loss of ogt-1. These results imply that GATA factors are able to activate OGT expression, which could be beneficial for longevity and the development of therapeutic treatment for aging-related diseases.
    Keywords:  Caenorhabditis elegans; GATA factor ELT-2; OGT-1; lifespan
  28. Int J Mol Sci. 2020 Jul 06. pii: E4777. [Epub ahead of print]21(13):
    He F, Ru X, Wen T.
      Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that regulates the cellular defense against toxic and oxidative insults through the expression of genes involved in oxidative stress response and drug detoxification. NRF2 activation renders cells resistant to chemical carcinogens and inflammatory challenges. In addition to antioxidant responses, NRF2 is involved in many other cellular processes, including metabolism and inflammation, and its functions are beyond the originally envisioned. NRF2 activity is tightly regulated through a complex transcriptional and post-translational network that enables it to orchestrate the cell's response and adaptation to various pathological stressors for the homeostasis maintenance. Elevated or decreased NRF2 activity by pharmacological and genetic manipulations of NRF2 activation is associated with many metabolism- or inflammation-related diseases. Emerging evidence shows that NRF2 lies at the center of a complex regulatory network and establishes NRF2 as a truly pleiotropic transcription factor. Here we summarize the complex regulatory network of NRF2 activity and its roles in metabolic reprogramming, unfolded protein response, proteostasis, autophagy, mitochondrial biogenesis, inflammation, and immunity.
    Keywords:  NRF2; UPR; autophagy; inflammation; metabolism; oxidative stress; proteostasis; transcription factor
  29. Mol Psychiatry. 2020 Jul 07.
    Pérez MJ, Ivanyuk D, Panagiotakopoulou V, Di Napoli G, Kalb S, Brunetti D, Al-Shaana R, Kaeser SA, Fraschka SA, Jucker M, Zeviani M, Viscomi C, Deleidi M.
      Mutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressing syndrome characterized by cerebellar ataxia, psychotic episodes, and obsessive behavior, as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1-knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPRmt) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid β in PITRM1-knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the other hand, over time, cerebral organoids generated from PITRM1-knockout iPSCs spontaneously developed pathological features of Alzheimer's disease (AD), including the accumulation of protein aggregates, tau pathology, and neuronal cell death. Single-cell RNA sequencing revealed a perturbation of mitochondrial function in all cell types in PITRM1-knockout cerebral organoids, whereas immune transcriptional signatures were substantially dysregulated in astrocytes. Importantly, we provide evidence of a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. Here, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPRmt that, in turn, modulates cytosolic quality control pathways. Thus, our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.
  30. Cell Rep. 2020 Jul 07. pii: S2211-1247(20)30827-5. [Epub ahead of print]32(1): 107846
    Kobiita A, Godbersen S, Araldi E, Ghoshdastider U, Schmid MW, Spinas G, Moch H, Stoffel M.
      The ability of pancreatic β-cells to respond to increased demands for insulin during metabolic stress critically depends on proper ribosome homeostasis and function. Excessive and long-lasting stimulation of insulin secretion can elicit endoplasmic reticulum (ER) stress, unfolded protein response, and β-cell apoptosis. Here we show that the diabetes susceptibility gene JAZF1 is a key transcriptional regulator of ribosome biogenesis, global protein, and insulin translation. JAZF1 is excluded from the nucleus, and its expression levels are reduced upon metabolic stress and in diabetes. Genetic deletion of Jazf1 results in global impairment of protein synthesis that is mediated by defects in ribosomal protein synthesis, ribosomal RNA processing, and aminoacyl-synthetase expression, thereby inducing ER stress and increasing β-cell susceptibility to apoptosis. Importantly, JAZF1 function and its pleiotropic actions are impaired in islets of murine T2D and in human islets exposed to metabolic stress. Our study identifies JAZF1 as a central mediator of metabolic stress in β-cells.
    Keywords:  ER stress; Jazf1; aminoacyl-tRNA synthetase; apoptosis; diabetes; insulin; rRNA processing; ribosomal proteins; ribosome biogenesis; transcription
  31. Nature. 2020 Jul 08.
    Giraldo MI, Xia H, Aguilera-Aguirre L, Hage A, van Tol S, Shan C, Xie X, Sturdevant GL, Robertson SJ, McNally KL, Meade-White K, Azar SR, Rossi SL, Maury W, Woodson M, Ramage H, Johnson JR, Krogan NJ, Morais MC, Best SM, Shi PY, Rajsbaum R.
      Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.
  32. FEBS J. 2020 Jul 10.
    Ferdosh S, Banerjee S, Pathak BK, Sengupta J, Barat C.
      Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starvation and the stationary phase of bacterial cell growth. Translational suppression involves the formation of either factor-bound inactive 70S monomers or dimeric 100S hibernating ribosomal complexes, the biological significance of which is poorly understood. Here, we demonstrate that the E. coli 70S ribosome associated with stationary phase factors HPF or YfiA and the 100S ribosome isolated from both gram-negative and gram-positive bacteria are resistant to unfolded protein-mediated subunit dissociation and subsequent degradation by cellular ribonucleases. Considering that the increase in cellular stress is accompanied by accumulation of unfolded proteins, such resistance of hibernating ribosomes towards dissociation might contribute to their maintenance during the stationary phase. Analysis of existing structures provided clues on the mechanism of inhibition of the unfolded protein-mediated disassembly in case of hibernating factor-bound ribosome. Further, the factor-bound 70S and 100S ribosomes can suppress protein aggregation and assist in protein folding. The chaperoning activity of these ribosomes is the first evidence of a potential biological activity of the hibernating ribosome that might be crucial for cell survival under stress conditions.
    Keywords:  Chaperoning activity; Hibernating ribosomes; Ribosome degradation; Stationary phase; Unfolded protein-mediated dissociation
  33. Nature. 2020 Jul 08.
    Gallotta I, Sandhu A, Peters M, Haslbeck M, Jung R, Agilkaya S, Blersch JL, Rödelsperger C, Röseler W, Huang C, Sommer RJ, David DC.
      In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1-5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6-9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.
  34. Curr Biol. 2020 Jul 06. pii: S0960-9822(20)30666-7. [Epub ahead of print]30(13): R770-R773
    Roberts MA, Segura-Roman A, Olzmann JA.
      Lipid droplets (LDs) are neutral lipid storage organelles assembled at the endoplasmic reticulum (ER). A new study reveals that the high membrane curvature of ER tubules catalyzes the nucleation of a neutral lipid lens, an early step in LD biogenesis.
  35. Autophagy. 2020 Jul 05. 1-21
    Sanchez-Garrido J, Shenoy AR.
      Nutrients not only act as building blocks but also as signaling molecules. Nutrient-availability promotes cell growth and proliferation and suppresses catabolic processes, such as macroautophagy/autophagy. These effects are mediated by checkpoint kinases such as MTOR (mechanistic target of rapamycin kinase), which is activated by amino acids and growth factors, and AMP-activated protein kinase (AMPK), which is activated by low levels of glucose or ATP. These kinases have wide-ranging activities that can be co-opted by immune cells upon exposure to danger signals, cytokines or pathogens. Here, we discuss recent insight into the regulation and repurposing of nutrient-sensing responses by the innate immune system during infection. Moreover, we examine how natural mutations and pathogen-mediated interventions can alter the balance between anabolic and autophagic pathways leading to a breakdown in tissue homeostasis and/or host defense.ABBREVIATIONS: AKT1/PKB: AKT serine/threonine kinase 1; ATG: autophagy related; BECN1: beclin 1; CGAS: cyclic GMP-AMP synthase; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; FFAR: free fatty acid receptor; GABARAP: GABA type A receptor-associated protein; IFN: interferon; IL: interleukin; LAP: LC3-associated phagocytosis; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NLR: NOD (nucleotide-binding oligomerization domain) and leucine-rich repeat containing proteins; PI3K, phosphoinositide 3-kinase; PRR: pattern-recognition receptor; PtdIns3K: phosphatidylinositol 3-kinase; RALB: RAS like proto-oncogene B; RHEB: Ras homolog, MTORC1 binding; RIPK1: receptor interacting serine/threonine kinase 1; RRAG: Ras related GTP binding; SQSTM1/p62: sequestosome 1; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; STK11/LKB1: serine/threonine kinase 11; TBK1: TANK binding kinase 1; TLR: toll like receptor; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; TRIM: tripartite motif protein; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H+-proton-translocating ATPase.
    Keywords:  AMPK; LC3-associated phagocytosis; MTOR; immunity; microbial pathogenesis; unconventional secretion
  36. EMBO Rep. 2020 Jul 09. e49898
    Zhang J, Andersen JP, Sun H, Liu X, Sonenberg N, Nie J, Shi Y.
      Nutrient sensing by the mTOR complex 1 (mTORC1) requires its translocation to the lysosomal membrane. Upon amino acids removal, mTORC1 becomes cytosolic and inactive, yet its precise subcellular localization and the mechanism of inhibition remain elusive. Here, we identified Aster-C as a negative regulator of mTORC1 signaling. Aster-C earmarked a special rough ER subdomain where it sequestered mTOR together with the GATOR2 complex to prevent mTORC1 activation during nutrient starvation. Amino acids stimulated rapid disassociation of mTORC1 from Aster-C concurrently with assembly of COP I vesicles which escorted mTORC1 to the lysosomal membrane. Consequently, ablation of Aster-C led to spontaneous activation of mTORC1 and dissociation of TSC2 from lysosomes, whereas inhibition of COP I vesicle biogenesis or actin dynamics prevented mTORC1 activation. Together, these findings identified Aster-C as a missing link between lysosomal trafficking and mTORC1 activation by revealing an unexpected role of COP I vesicles in mTORC1 signaling.
    Keywords:  ARF1; COP I; GRAMD1C; lysosomes; mTORC1
  37. EMBO J. 2020 Jul 09. e104730
    Dong R, Libby KA, Blaeschke F, Fuchs W, Marson A, Vale RD, Su X.
      The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, nor actin remodeling following CAR activation. However, CAR T cells still require LAT for an optimal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.
    Keywords:   CAR ; LAT ; T cell signaling; actin; immunological synapse
  38. Aging (Albany NY). 2020 Jul 09. 12
    Zhong J, Ouyang H, Zheng S, Guo Z, Chen Y, Zhong Y, Zhong W, Zuo L, Lu J.
      Mitochondria and the endoplasmic reticulum (ER) are known to promote cardiac ischemia/reperfusion (I/R) injury. Overexpression of yes-associated protein (YAP) and/or sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) has been shown to protect cardiomyocytes against I/R-induced injury. Here, we show that activation of the YAP/SERCA2a pathway attenuated mitochondrial damage and ER stress (ERS) to maintain cardiomyocyte viability in the setting of I/R injury. Our results demonstrate that I/R treatment reduced the transcription and expression of YAP and SERCA2a, along with a decline in cardiomyocyte viability. The overexpression of YAP promoted SERCA2a transcription, whereas SERCA2a upregulation did not affect the YAP transcription, suggesting that YAP functions upstream of SERCA2a. Activation of the YAP/SERCA2a pathway suppressed mitochondrial damage by sustaining the mitochondrial redox balance and restoring mitochondrial bioenergetics. Additionally, its activation repressed ERS, reduced calcium overload, and eventually blocked caspase activation. The knockdown of SERCA2a suppressed the protective effects of YAP overexpression on mitochondrial damage and ERS. Overall, our findings reveal that the YAP/SERCA2a pathway attenuates the mitochondrial damage and ERS in response to cardiac I/R injury by regulating the mitochondria-ER communication.
    Keywords:  ER; I/R injury; SERCA2a; YAP; cardiomyocytes; mitochondrial
  39. iScience. 2020 Jun 20. pii: S2589-0042(20)30438-7. [Epub ahead of print]23(7): 101252
    Wang T, Wei Q, Liang L, Tang X, Yao J, Lu Y, Qu Y, Chen Z, Xing G, Cao X.
      The accumulation of giant lipid droplets (LDs) increases the risk of metabolic disorders including obesity and insulin resistance. The lipolysis process involves the activation and transfer of lipase, but the molecular mechanism is not completely understood. The translocation of ATGL, a critical lipolysis lipase, from the ER to the LD surface is mediated by an energy catabolism complex. Oxysterol-binding protein-like 2 (OSBPL2/ORP2) is one of the lipid transfer proteins that regulates intracellular cholesterol homeostasis. A recent study has proven that Osbpl2-/- pigs exhibit hypercholesterolemia and obesity phenotypes with an increase in adipocytes. In this study, we identified that OSBPL2 links the endoplasmic reticulum (ER) with LDs, binds to COPB1, and mediates ATGL transport. We provide important insights into the function of OSBPL2, indicating that it is required for the regulation of lipid droplet lipolysis.
    Keywords:  Biological Sciences; Cell Biology; Molecular Biology
  40. Nat Chem. 2020 Jul 06.
    Gulen B, Rosselin M, Fauser J, Albers MF, Pett C, Krisp C, Pogenberg V, Schlüter H, Hedberg C, Itzen A.
      Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.
  41. Neuron. 2020 Jun 26. pii: S0896-6273(20)30469-4. [Epub ahead of print]
    Liu X, Beaudoin JD, Davison CA, Kosmaczewski SG, Meyer BI, Giraldez AJ, Hammarlund M.
      The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) essential for axon regeneration in Caenorhabditis elegans. We show that the xbp-1 ncRNA acts independently of the protein-coding function of the xbp-1 transcript as part of a dual output xbp-1 mRNA stress response axis. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding, but not the coding, function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.
    Keywords:  C. elegans; RNA processing; XBP1; axon regeneration; ncRNA; neuronal injury response; non-coding RNA; xbp-1
  42. Mol Cell Biol. 2020 Jul 06. pii: MCB.00122-20. [Epub ahead of print]
    Osei-Amponsa V, Sridharan V, Tandon M, Evans CN, Klarmann K, Cheng KT, Lack J, Chari R, Walters KJ.
      hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding, but defective for proteasome interaction. trRpn13 cells demonstrates reduced levels of proteasome-bound ubiquitinated proteins, indicating that loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicate that loss of full-length hRpn13 causes corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but that hRpn11 is elevated in ΔhRpn13 and trRpn13, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anti-cancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.
  43. Aging Cell. 2020 Jul 06. e13187
    Liang W, Moyzis AG, Lampert MA, Diao RY, Najor RH, Gustafsson ÅB.
      Advancing age is a major risk factor for developing heart disease, and the biological processes contributing to aging are currently under intense investigation. Autophagy is an important cellular quality control mechanism that is reduced in tissues with age but the molecular mechanisms underlying the age-associated defects in autophagy remain poorly characterized. Here, we have investigated how the autophagic process is altered in aged mouse hearts. We report that autophagic activity is reduced in aged hearts due to a reduction in autophagosome formation. Gene expression profile analysis to evaluate changes in autophagy regulators uncovered a reduction in Atg9b transcript and protein levels. Atg9 proteins are critical in delivering membrane to the growing autophagosome, and siRNA knockdown of Atg9b in cells confirmed a reduction in autophagosome formation. Autophagy is also the main pathway involved in eliminating dysfunctional mitochondria via a process known as mitophagy. The E3 ubiquitin ligase Parkin plays a key role in labeling mitochondria for mitophagy. We also found increased levels of Parkin-positive mitochondria in the aged hearts, an indication that they have been labeled for mitophagy. In contrast, Nrf1, a major transcriptional regulator of mitochondrial biogenesis, was significantly reduced in aged hearts. Additionally, our data showed reduced Drp1-mediated mitochondrial fission and formation of enlarged mitochondria in the aged heart. Overall, our findings suggest that cardiac aging is associated with reduced autophagosome number, decreased mitochondrial turnover, and formation of megamitochondria.
    Keywords:  Atg9; Parkin; aging; autophagy; heart; mitochondria; mitophagy
  44. Free Radic Biol Med. 2020 Jul 07. pii: S0891-5849(20)31107-2. [Epub ahead of print]
    Bao Y, Tong L, Song B, Liu G, Zhu Q, Lu X, Zhang J, Lu Y, Wen H, Tian Y, Sun Y, Zhu WG.
      Cancer therapeutics produce reactive oxygen species (ROS) that damage the cancer genome and lead to cell death. However, cancer cells can resist ROS-induced cytotoxicity and survive. We show that nuclear-localized uracil-DNA N-glycosylase isoform 2 (UNG2) has a critical role in preventing ROS-induced DNA damage and enabling cancer-cell resistance. Under physiological conditions, UNG2 is targeted for rapid degradation via an interaction with the E3 ligase UHRF1. In response to ROS, however, UNG2 protein in cancer cells exhibits a remarkably extended half-life. Upon ROS exposure, UNG2 is deacetylated at lysine 78 by histone deacetylases, which prevents the UNG2-UHRF1 interaction. Accumulated UNG2 protein can thus excise the base damaged by ROS and enable the cell to survive these otherwise toxic conditions. Consequently, combining HDAC inhibitors (to permit UNG2 degradation) with genotoxic agents (to produce cytotoxic cellular levels of ROS) leads to a robust synergistic killing effect in cancer cells in vitro. Altogether, these data support the application of a novel approach to cancer treatment based on promoting UNG2 degradation by altering its acetylation status using an HDAC inhibitor.
    Keywords:  HDAC inhibitor; Oxidative DNA damage; ROS; UHRF1; UNG2
  45. Cell Death Dis. 2020 Jul 08. 11(7): 513
    Feng Y, Zhong X, Tang TT, Wang C, Wang LT, Li ZL, Ni HF, Wang B, Wu M, Liu D, Liu H, Tang RN, Liu BC, Lv LL.
      Exosomes are increasingly recognized as vehicles of intercellular communication. However, the role of exosome in maintaining cellular homeostasis under stress conditions remained unclear. Here we show that Rab27a expression was upregulated exclusively in tubular epithelial cells (TECs) during proteinuria nephropathy established by adriamycin (ADR) injection and 5/6 nephrectomy as well as in chronic kidney disease patients, leading to the increased secretion of exosomes carrying albumin. The active exosome production promoted tubule injury and inflammation in neighboring and the producing cells. Interferon regulatory factor 1 (IRF-1) was found as the transcription factor contributed to the upregulation of Rab27a. Albumin could be detected in exosome fraction and co-localized with exosome marker CD63 indicating the secretion of albumin into extracellular space by exosomes. Interestingly, inhibition of exosome release accelerated albumin degradation which reversed tubule injury with albumin overload, while lysosome suppression augmented exosome secretion and tubule inflammation. Our findings revealed that IRF-1/Rab27a mediated exosome secretion constituted a coordinated approach to lysosome degradation for albumin handling, which lead to the augment of albumin toxicity as a maladaptive response to maintain cell homeostasis. The findings may suggest a novel therapeutic strategy for proteinuric kidney disease by targeting exosome secretion.
  46. Sci Rep. 2020 Jul 09. 10(1): 11327
    Herath V, Gayral M, Adhikari N, Miller R, Verchot J.
      The endoplasmic reticulum (ER) immunoglobulin binding proteins (BiPs) are molecular chaperones involved in normal protein maturation and refolding malformed proteins through the unfolded protein response (UPR). Plant BiPs belong to a multi-gene family contributing to development, immunity, and responses to environmental stresses. This study identified three BiP homologs in the Solanum tuberosum (potato) genome using phylogenetic, amino acid sequence, 3-D protein modeling, and gene structure analysis. These analyses revealed that StBiP1 and StBiP2 grouped with AtBiP2, whereas StBiP3 grouped with AtBiP3. While the protein sequences and folding structures are highly similar, these StBiPs are distinguishable by their expression patterns in different tissues and in response to environmental stressors such as treatment with heat, chemicals, or virus elicitors of UPR. Ab initio promoter analysis revealed that potato and Arabidopsis BiP1 and BiP2 promoters were highly enriched with cis-regulatory elements (CREs) linked to developmental processes, whereas BiP3 promoters were enriched with stress related CREs. The frequency and linear distribution of these CREs produced two phylogenetic branches that further resolve the groups identified through gene phylogeny and exon/intron phase analysis. These data reveal that the CRE architecture of BiP promoters potentially define their spatio-temporal expression patterns under developmental and stress related cues.
  47. Biochim Biophys Acta Mol Cell Res. 2020 Jul 06. pii: S0167-4889(20)30151-8. [Epub ahead of print] 118793
    Kandel RR, Neal SE.
      Cells are equipped with protein quality control pathways in order to maintain a healthy proteome; a process known as protein homeostasis. Dysfunction in protein homeostasis leads to the development of many diseases that are associated with proteinopathies. Recently, the rhomboid superfamily has attracted much attention concerning their involvement in protein homeostasis. While their functional role has become much clearer in the last few years, their systemic significance in mammals remains elusive. Here we delineate the current knowledge of rhomboids in protein quality control and how these functions are integrated at the organismal level.
    Keywords:  Derlins; ERAD; Protein homeostasis; Rhomboid protease; Rhomboid pseudoprotease