Defective targeting of hemojuvelin to plasma membrane is a common pathogenetic mechanism in juvenile hemochromatosis

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4503-4510 ◽  
Author(s):  
Laura Silvestri ◽  
Alessia Pagani ◽  
Claudia Fazi ◽  
Gianmario Gerardi ◽  
Sonia Levi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Iron Supplementation ◽  
Proteolytic Processing ◽  
Dual Function ◽  
Wild Type ◽  
Pathogenetic Mechanism ◽  
Gpi Anchor ◽  
Juvenile Hemochromatosis ◽  
Deficient Mice

Abstract Hemojuvelin (HJV) positively modulates the iron regulator hepcidin, and its mutations are the major cause of juvenile hemochromatosis (JH), a recessive disease leading to iron overload. Defective HJV reduces hepcidin up-regulation both in humans and in Hjv-deficient mice. To investigate the JH pathogenesis and the functional properties of human HJV we studied the biosynthesis and maturation of 6 HJV pathogenic mutants in HeLa and HepG2 cells. We show that proteolytic processing is defective in mutants F170S, W191C, and G320V, but not in G99V and C119F. Moreover, we show that mutants G99V and C119F are targeted to the cell surface, while F170S, W191C, G320V, and R326X (lacking the glycosilphosphatidylinositol [GPI] anchor) are mainly retained in the endoplasmic reticulum, although all mutants are released as soluble forms (s-HJV) in a proportion that is modulated by iron supplementation. Membrane HJV (m-HJV) is mainly composed of the cleaved protein, and its level is increased by iron in wild-type (WT) mice but not in the mutants. Altogether, the data demonstrate that the loss of HJV membrane export is central to the pathogenesis of JH, and that HJV cleavage is essential for the export. The results support a dual function for s- and m-HJV in iron deficiency and overload, respectively.

Ultrastructure and development of urediospore ornamentation in Melampsora lini

1971 ◽  
Vol 49 (12) ◽  
pp. 2067-2073 ◽  
Author(s):  
L. J. Littlefield ◽  
C. E. Bracker
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Outer Surface ◽  
Spore Wall ◽  
Wall Material ◽  
Wild Type ◽  
Melampsora Lini ◽  
Inner Surface ◽  
External Position ◽  
Basal Portion

The urediospores of Melampsora lini (Ehrenb.) Lev. are echinulate, with spines ca. 1 μ long over their surface. The spines are electron-transparent, conical projections, with their basal portion embedded in the electron-dense spore wall. The entire spore, including the spines, is covered by a wrinkled pellicle ca. 150–200 Å thick. The spore wall consists of three recognizable layers in addition to the pellicle. Spines form initially as small deposits at the inner surface of the spore wall adjacent to the plasma membrane. Endoplasmic reticulum occurs close to the plasma membrane in localized areas near the base of spines. During development, the spore wall thickens, and the spines increase in size. Centripetal growth of the wall encases the spines in the wall material. The spines progressively assume a more external position in the spore wall and finally reside at the outer surface of the wall. A mutant strain with finely verrucose spores was compared to the wild type. The warts on the surface of the mutant spores are rounded, electron-dense structures ca. 0.2–0.4 μ high, in contrast to spines of the wild type. Their initiation near the inner surface of the spore wall and their eventual placement on the outer surface of the spore are similar to that of spines. The wall is thinner in mutant spores than in wild-type spores.

scholarly journals Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.

1991 ◽  
Vol 174 (6) ◽  
pp. 1629-1637 ◽  
Author(s):  
J H Cox ◽  
J R Bennink ◽  
J W Yewdell
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Antigen Presentation ◽  
Viral Proteins ◽  
Genetically Engineered ◽  
Class I ◽  
Wild Type ◽  
Histocompatibility Complex ◽  
Infected Cells ◽  
Lumenal Domain

The E3/19K glycoprotein of adenovirus functions to diminish recognition of adenovirus-infected cells by major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) by binding intracellular class I molecules and preventing them from reaching the plasma membrane. In the present study we have characterized the nature of the interaction between E3/19K and the H-2Kd (Kd) molecule. An E3/19K molecule genetically engineered to terminate six residues from its normal COOH terminus (delta E19), was found to associate with Kd in a manner indistinguishable from wild-type E3/19K. Unlike E3/19K, however, delta E19 was transported through the Golgi complex to the plasma membrane, where it could be detected biochemically and immunocytochemically using a monoclonal antibody specific for the lumenal domain of E3/19K. Importantly, delta E19 also differed from E3/19K in being unable to prevent the presentation of Kd-restricted viral proteins to CTLs. This is unlikely to be due to delta E19 having a lower avidity for Kd than E3/19K, since delta E19 was able to compete with E3/19K for Kd binding, both physically, and functionally in nullifying the E3/19K blockade of antigen presentation. These findings indicate that the ability of E3/19K to block antigen presentation is due solely to its ability to retain newly synthesized class I molecules in the endoplasmic reticulum.

scholarly journals 12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response

2012 ◽  
Vol 302 (6) ◽  
pp. E654-E665 ◽  
Author(s):  
Banumathi K. Cole ◽  
Norine S. Kuhn ◽  
Shamina M. Green-Mitchell ◽  
Kendall A. Leone ◽  
Rebekah M. Raab ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Er Stress ◽  
Pancreatic Islets ◽  
High Fat Diet ◽  
Wild Type ◽  
High Fat ◽  
Stress Markers ◽  
Deficient Mice

Central obesity is associated with chronic inflammation, insulin resistance, β-cell dysfunction, and endoplasmic reticulum (ER) stress. The 12/15-lipoxygenase enzyme (12/15-LO) promotes inflammation and insulin resistance in adipose and peripheral tissues. Given that obesity is associated with ER stress and 12/15-LO is expressed in adipose tissue, we determined whether 12/15-LO could mediate ER stress signals. Addition of 12/15-LO lipid products 12(S)-HETE and 12(S)-HPETE to differentiated 3T3-L1 adipocytes induced expression and activation of ER stress markers, including BiP, XBP-1, p-PERK, and p-IRE1α. The ER stress inducer, tunicamycin, upregulated ER stress markers in adipocytes with concomitant 12/15-LO activation. Addition of a 12/15-LO inhibitor, CDC, to tunicamycin-treated adipocytes attenuated the ER stress response. Furthermore, 12/15-LO-deficient adipocytes exhibited significantly decreased tunicamycin-induced ER stress. 12/15-LO action involves upregulation of interleukin-12 (IL-12) expression. Tunicamycin significantly upregulated IL-12p40 expression in adipocytes, and IL-12 addition increased ER stress gene expression; conversely, LSF, an IL-12 signaling inhibitor, and an IL-12p40-neutralizing antibody attenuated tunicamycin-induced ER stress. Isolated adipocytes and liver from 12/15-LO-deficient mice fed a high-fat diet revealed a decrease in spliced XBP-1 expression compared with wild-type C57BL/6 mice on a high-fat diet. Furthermore, pancreatic islets from 12/15-LO-deficient mice showed reduced high-fat diet-induced ER stress genes compared with wild-type mice. These data suggest that 12/15-LO activity participates in ER stress in adipocytes, pancreatic islets, and liver. Therefore, reduction of 12/15-LO activity or expression could provide a new therapeutic target to reduce ER stress and downstream inflammation linked to obesity.

scholarly journals Tetherin is an exosomal tether

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
James R Edgar ◽  
Paul T Manna ◽  
Shinichi Nishimura ◽  
George Banting ◽  
Margaret S Robinson
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Short Range ◽  
Wild Type ◽  
Concomitant Increase ◽  
Gpi Anchor ◽  
Fold Reduction ◽  
Cell Metastasis ◽  
Health And Disease ◽  
Prion Transmission

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions.

scholarly journals Subcellular distribution of selenium in deficient mouse liver

1989 ◽  
Vol 258 (2) ◽  
pp. 541-545 ◽  
Author(s):  
R Reiter ◽  
R Otter ◽  
A Wendel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Trace Element ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fast Response ◽  
Cell Compartment ◽  
Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

scholarly journals mCLCA4 ER processing and secretion requires luminal sorting motifs

2008 ◽  
Vol 295 (1) ◽  
pp. C279-C287 ◽  
Author(s):  
Chunlei Huan ◽  
Kai Su Greene ◽  
Bo Shui ◽  
Gwendolyn Spizz ◽  
Haitao Sun ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chloride Transport ◽  
Proteolytic Processing ◽  
Regulatory Sequences ◽  
Cellular Processing ◽  
Green Fluorescent

Ca+-activated Cl− channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH2- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

scholarly journals A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature

2011 ◽  
Vol 193 (2) ◽  
pp. 333-346 ◽  
Author(s):  
Matt West ◽  
Nesia Zurek ◽  
Andreas Hoenger ◽  
Gia K. Voeltz
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Curvature ◽  
3D Analysis ◽  
Wild Type ◽  
Nanometer Resolution ◽  
Ribosome Density ◽  
Multiple Stages

We analyzed the structure of yeast endoplasmic reticulum (ER) during six sequential stages of budding by electron tomography to reveal a three-dimensional portrait of ER organization during inheritance at a nanometer resolution. We have determined the distribution, dimensions, and ribosome densities of structurally distinct but continuous ER domains during multiple stages of budding with and without the tubule-shaping proteins, reticulons (Rtns) and Yop1. In wild-type cells, the peripheral ER contains cytoplasmic cisternae, many tubules, and a large plasma membrane (PM)–associated ER domain that consists of both tubules and fenestrated cisternae. In the absence of Rtn/Yop1, all three domains lose membrane curvature, ER ribosome density changes, and the amount of PM-associated ER increases dramatically. Deletion of Rtns/Yop1 does not, however, prevent bloated ER tubules from being pulled from the mother cisterna into the bud and strongly suggests that Rtns/Yop1 stabilize/maintain rather than generate membrane curvature at all peripheral ER domains in yeast.

scholarly journals The Functional Paradox of CD43 in Leukocyte Recruitment: A Study Using CD43-deficient Mice

1998 ◽  
Vol 188 (11) ◽  
pp. 2181-2186 ◽  
Author(s):  
Richard C. Woodman ◽  
Brent Johnston ◽  
Michael J. Hickey ◽  
Diane Teoh ◽  
Paul Reinhardt ◽  
...  
Keyword(s):  
Platelet Activating Factor ◽  
Flow Chamber ◽  
Cell Interactions ◽  
Dual Function ◽  
Wild Type ◽  
Flow Conditions ◽  
Deficient Mice

Although there is considerable evidence implicating a role for CD43 (leukosialin) in leukocyte cell–cell interactions, its precise function remains uncertain. Using CD43-deficient mice (CD43−/−) and intravital microscopy to directly visualize leukocyte interactions in vivo, we investigated the role of CD43 in leukocyte–endothelial cell interactions within the cremasteric microcirculation under flow conditions. Our studies demonstrated significantly enhanced leukocyte rolling and adhesion after chemotactic stimuli in CD43−/− mice compared with wild type mice. Using an in vitro flow chamber, we established that the enhanced rolling interactions of CD43−/− leukocytes, primarily neutrophils, were also observed using immobilized E-selectin as a substrate, suggesting that passive processes related to steric hindrance or charge repulsion were likely mechanisms. Despite increased adhesion and rolling interactions by CD43−/− leukocytes, we uncovered a previously unrecognized impairment of CD43−/− leukocytes to infiltrate tissues. Oyster glycogen–induced neutrophil and monocyte infiltration into the peritoneum was significantly reduced in CD43−/− mice. In response to platelet activating factor, CD43−/− leukocytes were impaired in their ability to emigrate out of the vasculature. These results suggest that leukocyte CD43 has a dual function in leukocyte–endothelial interactions. In addition to its role as a passive nonspecific functional barrier, CD43 also facilitates emigration of leukocytes into tissues.

scholarly journals Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice

2011 ◽  
Vol 300 (3) ◽  
pp. H892-H901 ◽  
Author(s):  
Barbara Baragatti ◽  
Enrica Ciofini ◽  
Francesca Scebba ◽  
Debora Angeloni ◽  
Daria Sodini ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Immunofluorescence Microscopy ◽  
Oxygen Sensing ◽  
Ductus Arteriosus ◽  
Wild Type ◽  
Phasic Response ◽  
Sensing System ◽  
Cytochrome P 450

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null ( Cyp3a−/−) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a−/− DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a −/− DA. However, responses of RA-treated WT and Cyp3a−/− mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a−/− newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.

Cofilin-1 – Induced Actin Reorganization and Phosphatidylserine Exposure in Platelets

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4153-4153 ◽  
Author(s):  
Swapan Dasgupta ◽  
Anhquyen Le ◽  
Sandra Haudek ◽  
Mark Entman ◽  
Perumal Thiagarajan
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Platelet Membrane ◽  
Procoagulant Activity ◽  
Actin Binding ◽  
Wild Type ◽  
Actin Reorganization ◽  
Cover Slip ◽  
A Cell ◽  
Deficient Mice

Abstract Background: Platelet activation leads to the transbilayer movement of phosphatidylserine (PS) from inner to the outer leaflet of membrane bilayer. Exteriorization of PS promotes platelet procoagulant activity by promoting the assembly of tenase and the prothrombinase complex on platelet membrane. In a previous study, we observed that Rho-associated coiled-coil kinase-1 (ROCK1) deficiency in murine platelets or ROCK inhibition by Y-27632 in human platelets resulted in increase in PS exposure and platelet procoagulant activity. ROCK1 deficient platelets had a marked decrease in phosphorylation of cofilin-1. Cofilin-1 decreases actin filament length by increasing the rate of dissociation of actin monomers and its activity is abolished by phosphorylation. These studies suggested a role for cofilin-1-induced actin reorganization in collagen-induced PS exposure. Cofilin-1 activity is also modulated by its interactions with phosphatidylinositol 4, 5-bisphosphate (PIP2) and a cofilin-1 binding protein, Wdr1, which enhances capacity of cofilin-1 to accelerate depolymerization by capping their barbed ends. Here, we studied the role of cofilin-1 phosphorylation and its interaction with PIP2 and Wdr1 in activation induced PS exposure in platelets. Materials and Methods: We isolated platelet membrane, cytosol and cytoskeleton through differential centrifugation from resting and collagen-stimulated platelets and assessed the relative abundance of cofilin-1 and phosphocofilin-1 by mobility shift in phosphate-affinity polyacrylamide gel electrophoresis. This method allows simultaneous detection of relative proportions of phosphoproteins and its nonphospho counterparts. For cofilin-1 and Wdr-1 distribution, platelets were immobilized on a polylysine-coated cover slip or on a collagen-coated cover slip, fixed, permeabilized and immunostained with appropriate antibody. In addition, PS exposure (FITC lactadherin binding), F-actin (Alexa Fluor 488-phalloidin) and calcium concentration (Fura-2AM fluorescence) were also measured. Results: Immunofluorescence images show that in resting platelets, cofilin-1 is present in distinct patches in the plasma membrane and following activation with collagen, cofilin-1 is redistributed in a discrete granular pattern throughout the cytoplasm. In parallel, we also studied the relative distribution and the phosphorylation status of cofilin-1 in various subcellular fractions of platelets. In resting platelets, cofilin-1 is present in the plasma membrane and in the cytosol but absent in the cytoskeleton. Following activation with collagen, cofilin-1 moves to the cytoskeleton with a concomitant decrease in the plasma membrane fraction. These results suggest that active cofilin-1 is incorporated into reorganizing actin cytoskeleton during platelet activation. In ROCK1-deficient mice, there is increased cofilin-1 in cytoskeletal fraction, which also correlates with increase in PS exposure. Blocking PIP2 hydrolysis by U73122 prevents cofilin-1 translocation and PS exposure. A cell permeable cofilin-1 peptide containing the actin binding site of cofilin-1 (amino acids Trp104 to Met115) at the carboxy terminus (that competitively inhibits cofilin-1 binding to F-actin), blocks cofilin-1 translocation and decreases PS exposure. Platelets from a Wdr1-deficient mice which express less than 2% of wild-type protein in platelets, showed impaired collagen-induced PS exposure despite equivalent increase in intra platelet calcium compared to wild-type platelets. Conclusion: Our results show cofilin-1 trafficking from membranre to cytoskeleton and subsequent actin reorganization precedes PS exposure during platelet activation. In ROCK1-deficient platelets, there is increased cofilin-1 activity due to decreased serine-3 phosphorylation and it is associated with increased cofilin-1 translocation to actin filaments and enhanced PS exposure. In contrast, inhibiting cofilin-1 translocation, either by inhibiting PIP2 hydrolysis or by competitive inhibition by a cell permeable peptide, prevents cofilin-1 trafficking and decreases PS exposure. Furthermore, in Wdr1-deficient mouse platelets, which have diminished cofilin-1 activity, have impaired PS exposure during platelet activation. Disclosures No relevant conflicts of interest to declare.

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