Human HeLa cell lince SILAC - The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell
DISEASE(S): Malignant Neoplasm Of Ovary
SUBMITTER: 
Joon won Lee
LAB HEAD: Min Jae Lee
PROVIDER: PXD003813 | Pride | 2016-09-27
REPOSITORIES: pride
Dataset's files
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| 20150429_JW_F1.raw | Raw | |||
| 20150429_JW_F10.raw | Raw | |||
| 20150429_JW_F11.raw | Raw | |||
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| 20150429_JW_F2.raw | Raw |
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Publications
The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins.
Jiang Yanxialei Y Lee Jeeyoung J Lee Jung Hoon JH Lee Joon Won JW Kim Ji Hyeon JH Choi Won Hoon WH Yoo Young Dong YD Cha-Molstad Hyunjoo H Kim Bo Yeon BY Kwon Yong Tae YT Noh Sue Ah SA Kim Kwang Pyo KP Lee Min Jae MJ
Autophagy 20160825 11
The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of ...[more]
Similar Datasets
Project description:The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.
| S-EPMC5103369 | biostudies-literature
Project description:In the arginylation branch of the N-end rule pathway, unacetylated N-terminal destabilizing residues function as essential determinants of protein degradation signals (N-degron). Here, we show that a neurostimulant, para-chloroamphetamine (PCA), specifically inhibits the Arg/N-end rule pathway, delaying the degradation of its artificial and physiological substrates, including regulators of G protein signaling 4 (RGS4), in vitro and in cultured cells. In silico computational analysis indicated that PCA strongly interacts with both UBR box and ClpS box, which bind to type 1 and type 2 N-degrons, respectively. Moreover, intraperitoneal injection of PCA significantly stabilized endogenous RGS4 proteins in the whole mouse brain and, particularly, in the frontal cortex and hippocampus. Consistent with the role of RGS4 in G protein signaling, treatment with PCA impaired the activations of GPCR downstream effectors in N2A cells, phenocopying ATE1-null mutants. In addition, levels of pathological C-terminal fragments of TDP43 bearing N-degrons (Arg208-TDP25) were significantly elevated in the presence of PCA. Thus, our study identifies PCA as a potential tool to understand and modulate various pathological processes regulated by the Arg/N-end rule pathway, including neurodegenerative processes in FTLD-U and ALS.
| S-EPMC4161967 | biostudies-literature
Project description:In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants.
| S-EPMC3959200 | biostudies-literature
Project description:Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington's disease.
| S-EPMC4344198 | biostudies-literature
Project description:Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIAs) are analogues of the products of PI3K that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of 5 active PIAs and the PI3K inhibitor LY294002 (LY) was performed in non-small cell lung cancer (NSCLC) cells using high-density oligonucleotide arrays. Gene ontology analysis revealed genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, while genes involved in DNA replication, repair and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into 3 groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3 and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB) and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity. H157 cells were plated at 2x10^6 in T-75 flasks in RPMI medium 1640 containing 10% FBS and incubated for 24h. The media was then changed to RPMI medium 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM PIA6 dissolved in DMSO for 0h, 2h, 6h or 12h, and an equal volume of DMSO was added to control samples. For the PIA comparison, 10 microM PIAs (5, 6, 7, 23, 24, 25) or 10 microM LY294002 (LY) were incubated with the cells for 6h. In the PIA comparison experiment, the same concentration of PIA7 (an inert analog consisting of only the lipid side chain) was used as a control. Following incubation, the alterations in cellular morphology were photographed, and cells from 6-well plates were harvested for immunoblot analysis. Total RNA was extracted from cells treated in T-75 flasks using TRIzol reagent (Invitrogen) and chloroform, and purified according to the RNeasy midiprep spin kit protocol (Qiagen). Oligonucleotide microarray was performed with dye-swap. Microarray chips were generated from the 34,580 longmer probe set Human Genome Oligo Set Version 3.0 (Qiagen). Protocols for cDNA labeling, hybridization, and scanning are available through the National Human Genome Research Institute microarray core.
2011-03-22 | E-GEOD-27911 | biostudies-arrayexpress
Project description:Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy. H157 cells were plated 2 x 10^6 in T-75 flasks in RPMI 1640 containing 10% FBS and incubated for 24h. The medium was then changed to RPMI 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM of LY294002, OSU03012, PIA23, Perifosine, Miltefosine, API-2, DZ-50, or 100 nM of Wortmannin and Rapamycin for 6 hours, or an equal amount of DMSO as control. Following incubation, total RNA was extracted from cells in T-75 flasks using TRIzol reagent (Invitrogen). Two dye-swapped replicates were performed for each treatment.
2015-01-29 | E-GEOD-28993 | biostudies-arrayexpress
Project description:The similar characteristics shared by island environments have been shown to lead to common patterns of adaptations in island species, commonly referred to as the 'insularity syndrome'. A well-known example is the 'island rule', where large species become smaller on islands and small species become larger, leading to well-known cases of dwarfism and gigantism. This pattern was recently verified on a global scale, but the mechanisms underlying it have been poorly investigated. Here, we focused on the role of released pressure from predation and competition experienced by island birds. Using 120 pairs of endemic island species and their mainland sister relatives, we first verified that the island rule was detected in our dataset, and then evaluated the effects of the numbers of raptors and interspecific competitors on the evolution of the insular species' body mass. We found a strong effect of predation on body mass evolution, with a stronger island rule for species occurring on islands with no raptors, while the pattern disappears in their presence. However, we did not find an effect of competition on this pattern. Our study shows the importance of considering ecological interactions for understanding patterns of body size evolution, and the exceptions to those patterns.
| S-EPMC10031422 | biostudies-literature
Project description:Aerobic anoxygenic phototrophs: their role in carbon utilization under light in freshwater ecosystems
| PRJEB41596 | ENA
Project description:Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.
| S-EPMC8184782 | biostudies-literature
Project description:BackgroundAdvanced age is associated with both left bundle branch block (LBBB) and hypertension and the usefulness of ECG criteria to detect left ventricular hypertrophy (LVH) in patients with LBBB is still unclear. The diagnostic performance and clinical applicability of ECG-based LVH criteria in patients with LBBB defined by stricter ECG criteria is unknown. The aim of this study was to compare diagnostic accuracy and clinical utility of ECG criteria in patients with advanced age and strict LBBB criteria.MethodsRetrospective single-center study conducted from Jan/2017 to Mar/2018. Patients undergoing both ECG and echocardiogram examinations were included. Ten criteria for ECG-based LVH were compared using LVH defined by the echocardiogram as the gold standard. Sensitivity, specificity, predictive values, likelihood ratios, AUC, and the Brier score were used to compare diagnostic performance and a decision curve analysis was performed.ResultsFrom 4621 screened patients, 68 were included, median age was 78.4 years, (IQR 73.3-83.4), 73.5% with hypertension. All ECG criteria failed to provide accurate discrimination of LVH with AUC range between 0.54 and 0.67, and no ECG criteria had a balanced tradeoff between sensitivity and specificity. No ECG criteria consistently improved the net benefit compared to the strategy of performing routine echocardiogram in all patients in the decision curve analysis within the 10-60% probability threshold range.ConclusionECG-based criteria for LVH in patients with advanced age and true LBBB lack diagnostic accuracy or clinical usefulness and should not be routinely assessed.
| S-EPMC8600759 | biostudies-literature