91,458 research outputs found
The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi
No full textA procedure has been established in Vero cells for the isolation of an intermediate compartment involved in protein transport from the ER to the Golgi apparatus. The two-step subcellular fractionation procedure consists of Percoll followed by Metrizamide gradient centrifugation. Using the previously characterized p53 as a marker protein, the average enrichment factor of the intermediate compartment was 41. The purified fraction displayed a unique polypeptide pattern. It was largely separated from the rough ER proteins ribophorin I, ribophorin II, BIP, and protein disulfide isomerase, as well as from the putative cis-Golgi marker N-acetylglucosamine-1-phosphodiester-alpha-N-acetylglucosaminidase, the second of the two enzymes generating the lysosomal targeting signal mannose-6-phosphate. The first enzyme, N-acetylglucosaminylphosphotransferase, for which previous biochemical evidence had suggested both a pre- and a cis-Golgi localization in other cell types, cofractionated with the cis-Golgi rather than the intermediate compartment in Vero cells. The results suggest that the intermediate compartment defined by p53 has unique properties and does not exhibit typical features of rough ER and cis-Golgi
Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway
No full textERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant
Carbohydrate- and conformation-dependent cargo capture for ER-exit
No full textSome secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor
MYC and N-MYC are overexpressed in MYC-ER and N-MYC-ER-expressing cells, and naturally overexpressed MYC controls circadian gene expression.
No full textA.MYCN was determined in parental SKNAS, SKNAS N-MYC-ER, or SHEP N-MYC-ER by qPCR in n = 3 replicates by quantitative PCR (qPCR), normalized to β2M. MYCN overexpression is shown relative to parental SKNAS, since SHEP do not express MYCN. B. C-MYC-ER or endogenous c-MYC (endo) are shown in n = 3 immunoblot replicates, and quantitation is shown in the right panel, relative to Tubulin. C. The PC3 prostate cancer cell line, known to express high MYC, was treated with 6 μM of the MYC inhibitor MYCi361 for 24 hours in biological triplicates, as previously published [41]. Raw RNA-sequencing data was downloaded and processed (see Methods), and Deseq2 was used to compare cells ± MYCi361. Log2FC of indicated genes is shown. For A,B, error bars are standard error of the mean (S.E.M.) and ** is p C, error bars are Standard Error as calculated by DeSeq2, and * indicates padj (TIFF)</p
Er-doped Oxidized Porous Silicon Waveguides
No full textThe present work reports Er-doped channel oxidized porous silicon waveguides (OPSWG) formed from n+-type Si by the two-step anodisation process. Er has been introduced into porous silicon before oxidation by a cathodic treatment in 0.1 M Er (NO3)3 aqueous solution. A correlation between Er concentration and refractive index profiles has shown dominant core doping with Er relative to cladding regions. Reported Er concentration of 0.8 at.% in the OPSWG is large enough to attain the amplification effect
A Role for Macro-ER-Phagy in ER Quality Control.
No full textThe endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20-50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole
The formation and function of ER-endosome membrane contact sites
No full textRecent advances in membrane contact site (MCS) biology have revealed key roles for MCSs in inter-organellar exchange, the importance of which is becoming increasingly apparent. Roles for MCSs in many essential physiological processes including lipid transfer, calcium exchange, receptor tyrosine kinase signalling, lipid droplet formation, autophagosome formation, organelle dynamics and neurite outgrowth have been reported. The ER forms an extensive and dynamic network of MCSs with a diverse range of functionally distinct organelles. MCSs between the ER and endocytic pathway are particularly abundant, suggesting important physiological roles. Here, our current knowledge of the formation and function of ER contact sites with endocytic organelles from studies in mammalian systems is reviewed. Their relatively poorly defined molecular composition and recently identified functions are discussed. In addition, likely, but yet to be established, roles for these contacts in lipid transfer and calcium signalling are considered. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim Levine and Anant K. Menon
INTERICTAL ACTIVITY AFFECTS CORTICAL FFA AND DAG LEVELS DURING RECURRENT SEIZURES IN RATS
No full textGedragsinzichten bieden meer beleidskansen dan er nu worden benut
No full textGedragsinzichten zijn onontbeerlijk om te komen tot effectief beleid, de mens is immers geen homo economicus. Dat geldt echter niet alleen voor uitvoeringsvraagstukken, maar ook voor het ontwerpen van beleid. Hier ligt er nog een groot onbenut potentieel.Policy Analysi
DataSheet_1_Andrographolide Inhibits ER-Positive Breast Cancer Growth and Enhances Fulvestrant Efficacy via ROS-FOXM1-ER-α Axis.pdf
No full textEstrogen receptor (ER)-positive breast cancer is the main subtype of breast cancer (BRCA) with high incidence and mortality. Andrographolide (AD), a major active component derived from the traditional Chinese medicine Andrographis paniculate, has substantial anti-cancer effect in various tumors. However, the antitumor efficacy and the underlying molecular mechanisms of AD on ER-positive breast cancer are poorly understood. In the present study, we demonstrated that andrographolide (AD) significantly inhibited the growth of ER-positive breast cancer cells. Mechanistically, AD suppressed estrogen receptor 1 (ESR1, encodes ER-α) transcription to inhibit tumor growth. Further studies revealed that AD induced ROS production to down-regulate FOXM1-ER-α axis. Conversely, inhibiting ROS production with N-acetylcysteine (NAC) elevated AD-decreased ER-α expression, which could be alleviated by FOXM1 knockdown. In addition, AD in combination with fulvestrant (FUL) synergistically down-regulated ER-α expression to inhibit ER-positive breast cancer both in vitro and in vivo. These findings collectively indicate that AD suppresses ESR1 transcription through ROS-FOXM1 axis to inhibit ER-positive breast cancer growth and suggest that AD might be a potential therapeutic agent and fulvestrant sensitizer for ER-positive breast cancer treatment.</p
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