Grp78, Grp94, and Grp170 interact with α1-antitrypsin mutants that are retained in the endoplasmic reticulum

2005 ◽  
Vol 289 (3) ◽  
pp. G444-G455 ◽  
Author(s):  
Bela Z. Schmidt ◽  
David H. Perlmutter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cellular Response ◽  
Gradient Centrifugation ◽  
Calcium Ionophore ◽  
Genetically Engineered ◽  
Cross Linking ◽  
Ionophore A23187 ◽  
Mutant Form ◽  
Sucrose Density Gradient Centrifugation ◽  
Chemical Cross Linking

In α1-antitrypsin (α1-AT) deficiency, a mutant form of α1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant α1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The α1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which α1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant α1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of ∼110–130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic α1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the α1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that ∼85% of α1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and ∼15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of α1-AT Z as well as on its residual secretion.

scholarly journals Interactions between Newly Synthesized Glycoproteins, Calnexin and a Network of Resident Chaperones in the Endoplasmic Reticulum

1997 ◽  
Vol 136 (3) ◽  
pp. 555-565 ◽  
Author(s):  
Utpal Tatu ◽  
Ari Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary Cells ◽  
Gradient Centrifugation ◽  
Chinese Hamster ◽  
Cross Linking ◽  
Intact Cells ◽  
Ovary Cells ◽  
Influenza Hemagglutinin ◽  
Infected Cells ◽  
Chemical Cross Linking

Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5–8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5–5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.

scholarly journals Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 892
Author(s):  
Alexandre Augusto Pereira Firmino ◽  
Michal Gorka ◽  
Alexander Graf ◽  
Aleksandra Skirycz ◽  
Federico Martinez-Seidel ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Proteome Analysis ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytoplasmic Ribosome ◽  
Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

scholarly journals Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

Formation of Platelet Strings and Microthrombi in the Presence of ADAMTS13 Inhibitor Does Not Require P-Selectin or β3 Integrin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1782-1782
Author(s):  
Anil K. Chauhan ◽  
Denisa D. Wagner
Keyword(s):  
Calcium Ionophore ◽  
Genetically Engineered ◽  
Ionophore A23187 ◽  
Type I ◽  
Wild Type ◽  
Endothelial Surface ◽  
Genetically Engineered Mice ◽  
Β3 Integrin ◽  
Mean Time

Abstract VWF is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies respectively. VWF released from the storage granules upon cellular activation or stimulation with secretagogues are extremely large and known as Ultra-Large von Willebrand factor (UL-VWF). Such filaments were first shown by Dong and colleagues (Dong et. al., 2002) and platelets were seen to align as string of beads on these UL-VWF multimers. These platelet strings are then rapidly cleaved to smaller size by the metalloprotease ADAMTS-13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13). Recently, we have similarly shown that in Adamts13−/− mice platelet strings form and remain on stimulated endothelium in veins, where they wave in the blood stream for many minutes (Motto et. al., 2005). We have also shown that antibody to ADAMTS13 induces platelet strings in activated venules and transient thrombi formation in activated microvenules of wild type similar to Adamts13−/− mice (Chauhan et. al., 2006). The obvious question is what anchors the UL-VWF mulimers to the endothelial surface? In a flow chamber model, using polyclonal antibodies to P-selectin or soluble P-selectin, it was proposed that P-selectin anchors the UL-VWF multimers to the endothelial surface. The present study was designed to address the role of P-selectin and that of the platelet and endothelial integrin β3 in platelet string formation in vivo using intravital microscopy and genetically engineered mice. We demonstrate that in histamine activated venules, causing Weibel-Palade bodies secretion, P-selectin is not required for formation or retention of VWF platelet strings on endothelium in vivo. The mean time the platelet strings (~30–100 μm long) anchored onto the endothelium was 45 s. This duration was significantly longer than in P-selectin−/− mice infused with control Ig (P<0.001) and similar to WT mice treated with the inhibitory antibody. Similar to wild type, 45 s after topical superfusion of calcium ionophore A23187, platelet strings forming transient thrombi formation was seen in microvenules (25–30 μm in diameter) of P-selectin−/− mice when the ADAMTS13 activity was inhibited. Platelet strings were either not seen or were very short lived in the β3−/− mice infused with control Ig, whereas in the mice infused with anti- ADAMTS13 antibody platelet strings could be seen that anchored to the endothelium with a mean time of 50 s (P<0.0001). These observations show that platelet strings are not held by β3 integrin and platelets lacking β3 integrin can adhere firmly to the VWF strings in the histamine activated venules. VWF containing platelet strings could also entangle and form emboli in the absence of β3 integrin.

scholarly journals Localization of GRP78 to mitochondria under the unfolded protein response

2006 ◽  
Vol 396 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Fang-Chun Sun ◽  
Shou Wei ◽  
Chia-Wei Li ◽  
Yuo-Sheng Chang ◽  
Chih-Chung Chao ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Intermembrane Space ◽  
Atpase Inhibitor ◽  
Unfolded Protein ◽  
Stressed Cells ◽  
Protein Response ◽  
In Cells

The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results indicate that, at least under certain circumstances, the ER-resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signalling between these two organelles.

scholarly journals Competitive inhibition of a set of endoplasmic reticulum protein genes (GRP78, GRP94, and ERp72) retards cell growth and lowers viability after ionophore treatment.

1991 ◽  
Vol 11 (7) ◽  
pp. 3446-3453 ◽  
Author(s):  
X A Li ◽  
A S Lee
Keyword(s):  
Endoplasmic Reticulum ◽  
Competitive Inhibition ◽  
Chinese Hamster ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ovary Cell ◽  
Ionophore A23187 ◽  
Stress Protection ◽  
Endoplasmic Reticulum Protein ◽  
Ovary Cell Line

GRP78, a 78-kDa protein localized in the endoplasmic reticulum (ER), has been implicated in protein processing and stress protection. Its promoter contains a 36-bp region which is conserved among GRP genes across species and has the ability to compete for trans-acting factors mediating GRP gene expression. Integration of about 800 tandem copies of this sequence into the genome of a Chinese hamster ovary cell line (DG44) results in transfectants with the following phenotypes: (i) the induction level of GRP78 by the calcium ionophore A23187 and tunicamycin is reduced 4- and 2-fold, respectively, (ii) the induction levels of two other ER luminal protein genes, GRP94 and ERp72, are simultaneously down-regulated, (iii) the growth rate of these cells is half that of transfectants without the amplified sequence, and (iv) cell viability is decreased by 25-fold after A23187 treatment. These results provide new evidence that ERp72 shares common trans-acting regulatory factors with the GRP genes and that a reduction of this set of ER proteins correlates with lower viability after ionophore treatment.

scholarly journals Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm

1976 ◽  
Vol 154 (2) ◽  
pp. 491-499 ◽  
Author(s):  
L Bowden ◽  
J. M Lord
Keyword(s):  
Endoplasmic Reticulum ◽  
Castor Bean ◽  
Ricinus Communis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Radioactivity ◽  
Polypeptide Composition ◽  
Endosperm Tissue ◽  
Sucrose Density Gradient Centrifugation

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.

scholarly journals Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex

2018 ◽  
Vol 115 (42) ◽  
pp. E9792-E9801 ◽  
Author(s):  
Saikat Chowdhury ◽  
Chinatsu Otomo ◽  
Alexander Leitner ◽  
Kazuto Ohashi ◽  
Ruedi Aebersold ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Lipid Membranes ◽  
De Novo ◽  
Cross Linking ◽  
Double Membrane ◽  
Membrane Compartments ◽  
Biochemical Analyses ◽  
Chemical Cross Linking

Autophagy is an enigmatic cellular process in which double-membrane compartments, called “autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER–phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER–phagophore association and/or tethers vesicles to the ER–phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

Leukotriene Release from Neutrophils of Patients on Hemodialysis with Cellulose Membranes

1992 ◽  
Vol 15 (2) ◽  
pp. 84-88 ◽  
Author(s):  
A. Jörres ◽  
D. Jörres ◽  
G.M. Gahl ◽  
E. Schulz ◽  
A. Mahiout
Keyword(s):  
Gradient Centrifugation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dialysis Patients ◽  
Healthy Donors ◽  
Significant Difference ◽  
Before And After ◽  
Ficoll Density Gradient Centrifugation ◽  
Cellulose Membranes

The role of cytokines in patients with chronic renal failure is currently under investigation. We therefore studied the release of leukotriene B4 (LTB4) from polymorphonuclear leukocytes (PMN) in stable dialysis patients treated with two different cellulose membranes, Cuprophan and Hemophan®, a modified cellulose with less complement activation. Six patients were treated for four weeks with Cuprophan then switched to Hemophan® for another four weeks. Before and after the last treatment of each period, PMN were separated from 20 ml heparinized blood by FICOLL density gradient centrifugation. Portions of 5x106 PMN were resuspended in Hanks' buffer and stimulated for 5 minutes with calcium ionophore A23187 (5 /μg/ml). LTB4 in cell supernatants was determined by specific radioimmunoassay. PMN from dialysis patients before HD released significantly (p<0.01) more LTB4 than healthy donors. No significant difference between pre- and post-dialysis values was observed with Cuprophan or Hemophan® dialyzers. Our data suggest that the acute effects of blood membrane interaction with either complement activating or non-activating dialyzers do not lead to changes in post-dialysis leukotriene metabolism, but leukotriene production is enhanced chronically in dialysis patients.

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