scholarly journals Different modes of internalization of apoptotic alkyl-lysophospholipid and cell-rescuing lysophosphatidylcholine

2003 ◽  
Vol 374 (3) ◽  
pp. 747-753 ◽  
Author(s):  
Arnold H. van der LUIT ◽  
Marianne BUDDE ◽  
Marcel VERHEIJ ◽  
Wim J. van BLITTERSWIJK
Keyword(s):  
Lipid Rafts ◽  
De Novo ◽  
Structural Similarity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Chemotherapeutic Drugs ◽  
Alternative Substrate ◽  
High Structural Similarity ◽  
Additional Cell ◽  
Cell Membrane Level

The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541–39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.

scholarly journals APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain

2003 ◽  
Vol 163 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Yuzhi Chen ◽  
Wenyun Liu ◽  
Donna L. McPhie ◽  
Linda Hassinger ◽  
Rachael L. Neve
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Dna Synthesis ◽  
Lipid Rafts ◽  
Neuronal Apoptosis ◽  
Dominant Negative ◽  
Regulatory Subunit ◽  
Dominant Negative Mutant ◽  
Protein Levels ◽  
Induced Apoptosis

APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons (Chen, Y., D.L. McPhie, J. Hirschberg, and R.L. Neve. 2000. J. Biol. Chem. 275:8929–8935). We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a lipid-enriched fraction called lipid rafts. We show that coexpression of a peptide representing the domain of APP-BP1 that binds to APP, abolishes the ability of overexpressed APP or the V642I mutant of APP to cause neuronal apoptosis and DNA synthesis. A dominant negative mutant of the NEDD8 conjugating enzyme hUbc12, which participates in the ubiquitin-like pathway initiated by APP-BP1, blocks neuronal apoptosis caused by APP, APP(V642I), C31, or overexpression of APP-BP1. Neurons overexpressing APP or APP(V642I) show increased APP-BP1 protein levels in lipid rafts. A similar increase in APP-BP1 in lipid rafts is observed in the Alzheimer's disease brain hippocampus, but not in less-affected areas of Alzheimer's disease brain. This translocation of APP-BP1 to lipid rafts is accompanied by a change in the subcellular localization of the ubiquitin-like protein NEDD8, which is activated by APP-BP1.

scholarly journals Molecular mechanisms of invadopodium formation

2005 ◽  
Vol 168 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Hideki Yamaguchi ◽  
Mike Lorenz ◽  
Stephan Kempiak ◽  
Corina Sarmiento ◽  
Salvatore Coniglio ◽  
...  
Keyword(s):  
Rna Interference ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Egf Receptor ◽  
De Novo ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Periphery ◽  
Matrix Degradation ◽  
Degradation Activity

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

2012 ◽  
Vol 443 (2) ◽  
pp. 427-437 ◽  
Author(s):  
Heike Riegler ◽  
Thomas Herter ◽  
Irina Grishkovskaya ◽  
Anja Lude ◽  
Malgorzata Ryngajllo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
De Novo ◽  
Homo Sapiens ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Structural Similarity ◽  
Highly Active ◽  
Glycan Chain ◽  
High Structural Similarity ◽  
Protein Sequence Identity

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30–40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

scholarly journals Selective cytotoxicity of intracellular amyloid β peptide1–42 through p53 and Bax in cultured primary human neurons

2002 ◽  
Vol 156 (3) ◽  
pp. 519-529 ◽  
Author(s):  
Yan Zhang ◽  
Richard McLaughlin ◽  
Cynthia Goodyer ◽  
Andréa LeBlanc
Keyword(s):  
Cell Death ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Neuronal Cell Death ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Death Pathway ◽  
Human Neurons

Extracellular amyloid β peptides (Aβs) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Aβ1–42 accumulation before extracellular Aβ deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Aβ is toxic to human neurons. Microinjections of Aβ1–42 peptide or a cDNA-expressing cytosolic Aβ1–42 rapidly induces cell death of primary human neurons. In contrast, Aβ1–40, Aβ40–1, or Aβ42–1 peptides, and cDNAs expressing cytosolic Aβ1–40 or secreted Aβ1–42 and Aβ1–40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Aβ1–42 peptides is neurotoxic. The nonfibrillized and fibrillized Aβ1–42 peptides are equally toxic. In contrast, Aβ1–42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Aβ1–42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Aβ1–42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Aβ1–42 is selectively cytotoxic to human neurons through the p53–Bax cell death pathway.

scholarly journals High Cell Sensitivity toHelicobacter pyloriVacA Toxin Depends on a GPI-anchored Protein and is not Blocked by Inhibition of the Clathrin-mediated Pathway of Endocytosis

2000 ◽  
Vol 11 (11) ◽  
pp. 3897-3909 ◽  
Author(s):  
Vittorio Ricci ◽  
Antoine Galmiche ◽  
Anne Doye ◽  
Vittorio Necchi ◽  
Enrico Solcia ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Membrane Lipid ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Membrane Microdomains ◽  
Dominant Negative Mutant ◽  
High Cell ◽  
Uptake Mechanism ◽  
Cell Sensitivity ◽  
Negative Mutant

Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains (“lipid rafts”), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.

Semaphorin3A signaling controls Fas (CD95)-mediated apoptosis by promoting Fas translocation into lipid rafts

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2290-2299 ◽  
Author(s):  
Simona Moretti ◽  
Antonio Procopio ◽  
Raffaella Lazzarini ◽  
Maria Rita Rippo ◽  
Roberto Testa ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Signaling Mechanism ◽  
Agonistic Antibody ◽  
Neuropilin 1 ◽  
Lipid Raft Microdomains ◽  
Biologic Function ◽  
Necrosis Factor

Semaphorins and their receptors (plexins) have pleiotropic biologic functions, including regulation of immune responses. However, the role of these molecules inside the immune system and the signal transduction mechanism(s) they use are largely unknown. Here, we show that Semaphorin3A (Sema3A) triggers a proapoptotic program that sensitizes leukemic T cells to Fas (CD95)-mediated apoptosis. We found that Sema3A stimulation provoked Fas translocation into lipid raft microdomains before binding with agonistic antibody or FasL (CD95L). Disruption of lipid rafts reduced sensitivity to Fas-mediated apoptosis in the presence of Sema3A. Furthermore, we show that plexin-A1, together with Sema3A-binding neuropilin-1, was rapidly incorporated into membrane rafts after ligand stimulation, resulting in the transport of actin-linking proteins into Fas-enriched rafts. Cells expressing a dominant-negative mutant of plexin-A1 did not show Fas clustering and apoptosis on Sema3A/Fas costimulation. This work identifies a novel biologic function of semaphorins and presents an unexpected signaling mechanism linking semaphorin to the tumor necrosis factor family receptors.

scholarly journals The role of JUN in the regulation of PRKCC-mediated STAR expression and steroidogenesis in mouse Leydig cells

2008 ◽  
Vol 41 (5) ◽  
pp. 329-341 ◽  
Author(s):  
Pulak R Manna ◽  
Douglas M Stocco
Keyword(s):  
Leydig Cells ◽  
De Novo ◽  
Dna Interaction ◽  
Dominant Negative ◽  
Proximal Promoter ◽  
Dominant Negative Mutant ◽  
Mobility Shift ◽  
Steroid Synthesis ◽  
Progesterone Synthesis

Activator protein 1 (JUN) transcription factors (JUN and FOS) play critical roles in a wide variety of signaling processes including those in the protein kinase C (PRKCC) pathway, a pathway that is instrumental in the expression of the steroidogenic acute regulatory (STAR) protein. In the present study, we determined the functional involvement of one of the key JUN family members, JUN, in the regulation of PRKCC-dependent STAR expression and steroidogenesis. MA-10 mouse Leydig tumor cells treated with an activator of PRKCC, phorbol 12-myristate 13-acetate (PMA), demonstrated increases in the expression of the STAR and CYP11A1 proteins and progesterone synthesis, which coincided with the expression and phosphorylation of JUN (P-JUN). PMA was also capable of enhancing the cAMP analog, (Bu)2cAMP, which stimulated JUN, STAR, P-STAR and progesterone levels. The induction of Jun mRNA expression and steroid synthesis by PMA requires de novo protein synthesis. Chromatin immunoprecipitation studies revealed the association of P-JUN with the STAR proximal promoter and that PMA specifically enhanced in vivo P-JUN–DNA interaction. Electrophoretic mobility shift assays and reporter gene analyses demonstrated that JUN binds to the JUN motif (−81/−75 bp) in the STAR promoter, and that JUN–DNA-binding activity was highly correlated with the induction of JUN by PRKCC signaling. Overexpression of JUN increased the PMA-mediated transcription of the Star gene, an event markedly decreased by TAM-67, a dominant negative mutant of JUN. Targeted silencing of endogenous JUN, by small interfering RNA, was correlated with the repression of basal- and PMA-mediated STAR expression and progesterone synthesis. These findings describe the mechanisms by which JUN influences PRKCC signaling and provide additional and novel insight into the regulation of the steroidogenic machinery in mouse Leydig cells.

scholarly journals A Novel Intronic Pathogenic Variant in STAR With a Dominant Negative Mechanism Causes Attenuated Lipoid Congenital Adrenal Hyperplasia

2021 ◽  
Vol 9 ◽  
pp. 232470962110146
Author(s):  
Erin Finn ◽  
Kimberly Kripps ◽  
Christina Chambers ◽  
Michele Rapp ◽  
Naomi J. L. Meeks ◽  
...  
Keyword(s):  
Congenital Adrenal Hyperplasia ◽  
Autosomal Recessive ◽  
De Novo ◽  
Dominant Negative ◽  
Adrenal Hyperplasia ◽  
Primary Adrenal Insufficiency ◽  
Heterozygous Variant ◽  
Novel Variant ◽  
Autosomal Recessive Condition ◽  
Clinically Significant

Lipoid congenital adrenal hyperplasia (LCAH) is typically inherited as an autosomal recessive condition. There are 3 reports of individuals with a dominantly acting heterozygous variant leading to a clinically significant phenotype. We report a 46,XY child with a novel heterozygous intronic variant in STAR resulting in LCAH with an attenuated genital phenotype. The patient presented with neonatal hypoglycemia and had descended testes with a fused scrotum and small phallus. Evaluation revealed primary adrenal insufficiency with deficiencies of cortisol, aldosterone, and androgens. He was found to have a de novo heterozygous novel variant in STAR: c.65-2A>C. We report a case of a novel variant and review of other dominant mutations at the same position in the literature. Clinicians should be aware of the possibility of attenuated genital phenotypes of LCAH and the contribution of de novo variants in STAR at c.65-2 to the pathogenesis of that phenotype.

scholarly journals Microwave Imaging of Breast Skin Utilizing Elliptical UWB Antenna and Reverse Problems Algorithm

Micromachines ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 647
Author(s):  
Sameer Alani ◽  
Zahriladha Zakaria ◽  
Tale Saeidi ◽  
Asmala Ahmad ◽  
Muhammad Ali Imran ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Imaging System ◽  
Signal Transmission ◽  
Similarity Index ◽  
Structural Similarity ◽  
Microwave Imaging ◽  
Ultra Wideband ◽  
Uwb Antenna ◽  
System A ◽  
High Structural Similarity

Skin cancer is one of the most widespread and fast growing of all kinds of cancer since it affects the human body easily due to exposure to the Sun’s rays. Microwave imaging has shown better outcomes with higher resolution, faster processing time, mobility, and less cutter and artifact effects. A miniaturized elliptical ultra-wideband (UWB) antenna and its semi-spherical array arrangement were used for signal transmission and reception from the defected locations in the breast skin. Several conditions such as various arrays of three, six, and nine antenna elements, smaller tumor, multi-tumors, and skin on a larger breast sample of 30 cm were considered. To assess the ability of the system, a breast shape container with a diameter of 130 mm and height of 60 mm was 3D printed and then filled with fabricated skin and breast fat to perform the experimental investigation. An improved modified time-reversal algorithm (IMTR) was used to recreate 2D images of tumors with the smallest radius of 1.75 mm in any location within the breast skin. The reconstructed images using both simulated and experimental data verified that the system can be a reliable imaging system for skin cancer diagnosis having a high structural similarity index and resolution.

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