scholarly journals Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

2013 ◽  
Vol 24 (11) ◽  
pp. 1735-1748 ◽  
Author(s):  
Zoe G. Holloway ◽  
Antonio Velayos-Baeza ◽  
Gareth J. Howell ◽  
Clotilde Levecque ◽  
Sreenivasan Ponnambalam ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Copper Transporter ◽  
Caveolin 1 ◽  
Clathrin Adaptor ◽  
Partial Overlap ◽  
Golgi Network

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.

scholarly journals A Caveolin Dominant Negative Mutant Associates with Lipid Bodies and Induces Intracellular Cholesterol Imbalance

2001 ◽  
Vol 152 (5) ◽  
pp. 1057-1070 ◽  
Author(s):  
Albert Pol ◽  
Robert Luetterforst ◽  
Margaret Lindsay ◽  
Sanna Heino ◽  
Elina Ikonen ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Free Cholesterol ◽  
Neutral Lipids ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intracellular Accumulation ◽  
Caveolin 1 ◽  
Late Endosomes ◽  
Intracellular Cholesterol

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3DGV, specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1–3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cavDGV to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

scholarly journals Polarized sorting of the copper transporter ATP7B in neurons mediated by recognition of a dileucine signal by AP-1

2015 ◽  
Vol 26 (2) ◽  
pp. 218-228 ◽  
Author(s):  
Shweta Jain ◽  
Ginny G. Farías ◽  
Juan S. Bonifacino
Keyword(s):  
Hippocampal Neurons ◽  
Copper Metabolism ◽  
Cell Types ◽  
Dominant Negative ◽  
Copper Transporter ◽  
Polarized Cells ◽  
Neurocutaneous Disorder ◽  
Cytosolic Domains ◽  
Clathrin Adaptor ◽  
Golgi Network

Neurons are highly polarized cells having distinct somatodendritic and axonal domains. Here we report that polarized sorting of the Cu2+ transporter ATP7B and the vesicle-SNARE VAMP4 to the somatodendritic domain of rat hippocampal neurons is mediated by recognition of dileucine-based signals in the cytosolic domains of the proteins by the σ1 subunit of the clathrin adaptor AP-1. Under basal Cu2+ conditions, ATP7B was localized to the trans-Golgi network (TGN) and the plasma membrane of the soma and dendrites but not the axon. Mutation of a dileucine-based signal in ATP7B or overexpression of a dominant-negative σ1 mutant resulted in nonpolarized distribution of ATP7B between the somatodendritic and axonal domains. Furthermore, addition of high Cu2+ concentrations, previously shown to reduce ATP7B incorporation into AP-1–containing clathrin-coated vesicles, caused loss of TGN localization and somatodendritic polarity of ATP7B. These findings support the notion of AP-1 as an effector of polarized sorting in neurons and suggest that altered polarity of ATP7B in polarized cell types might contribute to abnormal copper metabolism in the MEDNIK syndrome, a neurocutaneous disorder caused by mutations in the σ1A subunit isoform of AP-1.

scholarly journals Dominant negative FADD/MORT1 inhibits the development of intestinal intraepithelial lymphocytes with a marked defect on CD8αα+TCRγδ+ T cells

2018 ◽  
Author(s):  
Xuerui Zhang ◽  
Lina Huo ◽  
Lulu Song ◽  
Zhaoqing Hu ◽  
Xinran Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Death Receptor ◽  
Adaptor Protein ◽  
Intraepithelial Lymphocytes ◽  
Protective Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intestinal Intraepithelial Lymphocytes ◽  
Induced Apoptosis

AbstractIntestinal intraepithelial lymphocytes are considered to be distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. Although the development and homing of IELs have been studied in some details, the factors controlling their homeostasis are incompletely understood. Here, we demonstrate that FADD, a classic adaptor protein required for death-receptor-induced apoptosis, is a critical regulator of the intestinal IEL development. The mice with a dominant negative mutant of FADD (FADD-DN) display a defective localized intestinal IELs with a marked defect on CD8αα+TCRγδ+ T cells. Since Lin- LPLs have been identified as precursors CP cells for CD8αα+ development, we analyzed lamina propria lymphocytes (LPLs) and found the massive accumulation of IL-7R-lin- LPLs in FADD-DN mice. IL-7 plays a differentiation inducing role in the development of intestinal IELs and its receptor IL-7R is a transcriptional target of Notch1. The level of Notch1 expression also showed very low in Lin- LPLs cells from FADD-DN mice compared with normal mice, indicating a possible molecular mechanism of FADD in the early IEL development. In addition, loss of γδ T-IELs induced by FADD-DN results in a worsening inflammation in murine DSS-induced colitis model, suggesting a protective role of FADD in the intestinal homeostasis.

scholarly journals Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant μ2 subunit and its effects on endocytosis

1999 ◽  
Vol 18 (9) ◽  
pp. 2489-2499 ◽  
Author(s):  
Alexandre Nesterov ◽  
Royston E. Carter ◽  
Tatiana Sorkina ◽  
Gordon N. Gill ◽  
Alexander Sorkin
Keyword(s):  
Receptor Binding ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Binding Function ◽  
Clathrin Adaptor ◽  
Negative Mutant

scholarly journals The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

2015 ◽  
Vol 26 (10) ◽  
pp. 1887-1900 ◽  
Author(s):  
Steven D. Garafalo ◽  
Eric S. Luth ◽  
Benjamin J. Moss ◽  
Michael I. Monteiro ◽  
Emily Malkin ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nerve Cord ◽  
Ventral Nerve Cord ◽  
Secretory Pathway ◽  
Adaptor Protein ◽  
Intracellular Compartments ◽  
Clathrin Adaptor ◽  
Strength And Plasticity

Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway.

scholarly journals Suppression of eNOS-derived superoxide by caveolin-1: a biopterin-dependent mechanism

2011 ◽  
Vol 301 (3) ◽  
pp. H903-H911 ◽  
Author(s):  
Kanchana Karuppiah ◽  
Lawrence J. Druhan ◽  
Chun-an Chen ◽  
Travis Smith ◽  
Jay L. Zweier ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Oxidase Activity ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Caveolin 1 ◽  
Calcium Calmodulin Dependent ◽  
Enos Uncoupling ◽  
Nadph Oxidation ◽  
Interfering Rna

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (BH4), NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH4 oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH4 depletion alone. In the current study, we investigated the effects of both BH4 depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH4 does not result in eNOS oxidase activity, whereas BH4 oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms, which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches, we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using small interfering RNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH4 oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH4 depletion increased eNOS-derived superoxide to 165% of that observed with BH4 oxidation. This study identifies a critical role of caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease-associated changes in caveolin-1 expression may contribute to endothelial dysfunction.

Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

2001 ◽  
Vol 114 (20) ◽  
pp. 3749-3757 ◽  
Author(s):  
Patrick Meraldi ◽  
Erich A. Nigg
Keyword(s):  
Protein Phosphatase ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Microtubule Depolymerization ◽  
Drug Induced ◽  
Microtubule Network ◽  
Similar Phenotype ◽  
Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

scholarly journals MITOCHONDRIA AS REVERSIBLE REGULATORS OF AGING ASSOCIATED SKIN WRINKLES AND HAIR LOSS IN MICE

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S395-S395
Author(s):  
Keshav K Singh
Keyword(s):  
Mouse Model ◽  
Hair Loss ◽  
Transgene Expression ◽  
Skin Aging ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dominant Negative Mutation ◽  
Polymerase Domain ◽  
Skin Wrinkles

Abstract To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

scholarly journals ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

2010 ◽  
Vol 299 (5) ◽  
pp. C1015-C1027 ◽  
Author(s):  
Corina M. Balut ◽  
Yajuan Gao ◽  
Sandra A. Murray ◽  
Patrick H. Thibodeau ◽  
Daniel C. Devor
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Close Association ◽  
Significant Inhibition ◽  
Dominant Negative ◽  
Human Embryonic Kidney Cells ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Interfering Rna

The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

scholarly journals Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

2007 ◽  
Vol 404 (2) ◽  
pp. 179-190 ◽  
Author(s):  
Mark Windheim ◽  
Christine Lang ◽  
Mark Peggie ◽  
Lorna A. Plater ◽  
Philip Cohen
Keyword(s):  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Muramyl Dipeptide ◽  
Dominant Negative ◽  
Signalling Pathways ◽  
Dominant Negative Mutant ◽  
Protein Kinase Activity ◽  
P38α Mapk

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2–RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1β (interleukin-1β) and TNFα (tumour necrosis factor-α). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-β-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38α MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38α MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1β and TNFα in these cells. Taken together, our results indicate that the MDP–NOD2/RIP2 and LPS (lipopolysaccharide)–TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.

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