scholarly journals Mutation in the CPC motif-containing 6th transmembrane domain affects intracellular localization, trafficking and coppertransport efficiency of ATP7Aprotein in mosaic mutant mice––an animal model of Menkes disease

Metallomics ◽  
2012 ◽  
Vol 4 (2) ◽  
pp. 197-204 ◽  
Author(s):  
Małgorzata Lenartowicz ◽  
Paweł Grzmil ◽  
Moneef Shoukier ◽  
Rafał Starzyński ◽  
Marcin Marciniak ◽  
...  
Keyword(s):  
Animal Model ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Menkes Disease ◽  
Mutant Mice

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

2011 ◽  
Vol 11 (1-2) ◽  
pp. 41-47 ◽  
Author(s):  
Małgorzata Lenartowicz ◽  
Rafał Starzyński ◽  
Krzysztof Wieczerzak ◽  
Wojciech Krzeptowski ◽  
Paweł Lipiński ◽  
...  
Keyword(s):  
Animal Model ◽  
Postnatal Development ◽  
Menkes Disease ◽  
Mutant Mice ◽  
Early Postnatal Development ◽  
Atp7a Gene

scholarly journals Haemolysis and Perturbations in the Systemic Iron Metabolism of Suckling, Copper-Deficient Mosaic Mutant Mice – An Animal Model of Menkes Disease

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e107641 ◽  
Author(s):  
Małgorzata Lenartowicz ◽  
Rafał R. Starzyński ◽  
Wojciech Krzeptowski ◽  
Paweł Grzmil ◽  
Aleksandra Bednarz ◽  
...  
Keyword(s):  
Animal Model ◽  
Iron Metabolism ◽  
Menkes Disease ◽  
Mutant Mice

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

scholarly journals A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2

1998 ◽  
Vol 72 (3) ◽  
pp. 2183-2191 ◽  
Author(s):  
Laurence Cocquerel ◽  
Jean-Christophe Meunier ◽  
André Pillez ◽  
Czeslaw Wychowski ◽  
Jean Dubuisson
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Native Form ◽  
Glycosyl Phosphatidylinositol ◽  
Er Retention ◽  
Er Targeting ◽  
Necessary And Sufficient

ABSTRACT The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

2001 ◽  
Vol 114 (17) ◽  
pp. 3115-3124 ◽  
Author(s):  
Kazuo Kasai ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Immunofluorescence Analysis ◽  
Attachment Protein ◽  
Transport Pathways ◽  
Sensitive Factor ◽  
Protein Receptors

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

scholarly journals Mice with targeted disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3217-3228 ◽  
Author(s):  
J.M. Goddard ◽  
M. Rossel ◽  
N.R. Manley ◽  
M.R. Capecchi
Keyword(s):  
Animal Model ◽  
Facial Nerve ◽  
Mutant Mice ◽  
Clinical Profile ◽  
Human Diseases ◽  
Motor Nucleus ◽  
Moebius Syndrome ◽  
Targeted Disruption ◽  
Adult Mice ◽  
Motor Component

Mice were generated with targeted disruptions in the hoxb-1 gene. Two separate mutations were created: the first disrupts only the homeodomain and the second inactivates the first exon as well as the homeodomain. The phenotypes associated with these two mutant alleles are indistinguishable in surviving adult mice. The predominant defect in these mutant mice is a failure to form the somatic motor component of the VIIth (facial) nerve, possibly through a failure to specify these neurons. The phenotype of hoxb-1 mutant homozygotes closely resembles features of the clinical profile associated with humans suffering from Bell's Palsy or Moebius Syndrome. These animals should therefore provide a useful animal model for these human diseases.

SB431542, An Inhibitor of TGF-β1 Activin Receptor-Like Kinases, Improves the Natural History of Myelofibrosis In Gata1low Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 462-462
Author(s):  
Maria Zingariello ◽  
Barbara Ghinassi ◽  
Rosa Alba Rana ◽  
Maria Verrucci ◽  
Fabrizio Martelli ◽  
...  
Keyword(s):  
Animal Model ◽  
Disease Progression ◽  
Renal Fibrosis ◽  
Bone Structure ◽  
Mutant Mice ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Activin Receptor ◽  
Cell Counts ◽  
Tgf Β1

Abstract Abstract 462 The marrow microenvironment in primary myelofibrosis and mouse models of the disease is characterized by increased levels of cytokines which regulate hematopoiesis including CXCL12, BMP4, VEGF and TGF-β. The observation that TGF-βnull myelofibrotic stem cells fail to transmit the disease by transplantation (Chagraoui et al, Blood 100:3495, 2002) has established an important role for TGF-β in disease development. Mice carrying the hypomorphic Gata1low mutation in which the enhancer that drives gene expression in megakaryocytes (MK) is deleted also develop myelofibrosis with age (Vannucchi et al, Blood 2002;100:1123). To clarify the role of TGF-β in development of myelofibrosis in the Gata1low mouse model, the levels of this factor in plasma and marrow of the mutant mice were measured. Gata1low mice express normal levels of TGF-β in plasma (1.8±0.7 vs 2.1±0.4 ng/mL) and levels of TGF-β mRNA (2.9±0.5 vs 1.5±0.3 arbitrary units, p<0.01) and protein (1.9±0.3 vs 0.86±0.2 ng/mL, p<0.01) only 2-times greater than normal in marrow. However, by immunoelectron microscopy, patchy TGF-β deposits associated with collagen fibers were observed in the marrow microenvironment of mutant mice (<3 vs >700 particles/field in wild-type and Gata1low marrow, respectively) indicating that fibrosis, by concentrating TGF-β locally, may contribute to disease progression also in Gata1low mice. To evaluate whether inhibition of TGF-β signaling would ameliorate myelofibrosis in this animal model, Gata1low mice were treated with SB431542 (C22H16N4O3, MW= 384.4), an inhibitor of TGF- β1/activin receptor-like kinases recently demonstrated to prevent renal fibrosis in mice (Petersen et al, Kidney Int 73:705, 2008.). Six males (7-9 months) and 6 females (12 months) were treated with SB431542 as described for renal fibrosis (see Figure). Equivalent numbers of mice treated with vehicle were used as control. Treatment was well tolerated (no deaths) and the SB43542-treated mice were easily recognized by being more active and with shinier coats. At the end of the 4th cycle, mice were sacrificed and analyzed for disease progression. The results were as follows: Blood: SB43542-treatment did not affect hematocrit levels (43.2±1.2 vs 41.3±0.9 in SB43542- and vehicle-treated mice, respectively), increased platelets numbers [0.34(±0.03)×106/μL vs 0.2(±0.009)×106/μL, p<0.01] but platelets remained larger than normal, reduced white blood cell counts [5.2(±0.19)×103/μL vs 6.5(±0.3)×103/μL, p<0.01] and frequency of poikilocytes (1 every 4–5 fields vs >4/field). Also, progenitor cell trafficking was not reduced (CD34posCD117pos cells: 1.45 vs 1.2% colony forming cells: 10.8±2.4 vs 8.0±0.1 CFC/μL). Marrow: Treatment increased total cell number [18.0(±0.7) ×106 vs 8.2(±0.4) ×106/femur, p<0.01] and frequency of erythroid cells (20.5±2.5 vs 13.2±0.7%, p<0.01) but not of MK (40.2±5.7 vs 38.7±0.9%) in the femur. However, fibrosis and microvessel density were reduced (Gomori-Silver and CD34 staining). Increased Mallory staining of bones was observed but the femur became resistant to fracture, suggesting that overall bone structure improved. Spleen: SB43542-treatment reduced spleen weight (0.2±0.6 vs 0.45±0.05 gr, p<0.01) and cell numbers [265(±30)×106 vs 385(±5)×106 cells, p<0.01]. Therefore, although the frequency of erythroid cells and MK in the organ remained high, overall hematopoiesis in spleen was reduced. Liver: SB43542-treatment restored the morphological appearance of the liver and reduced the frequency of MK (5.9±0.7 vs 20.4±4.7, p<0.01). These improvements were likely not due to an anti-inflammatory effect of the drug because parallel treatments with dexamethasone did not modify disease progression in Gata1low mice. Conclusion: SB43542-treatment reduced the myelofibrotic traits expressed by Gata1low mice, confirming that increased TGF-β1 levels play an important role in disease manifestations in this animal model. We have previously published that Aplidin treatment restores the hematopoietic stem cell properties of Gata1low mice (Verrucci et al, J Cell Physiol,2010, May20, Epub ahead of print). The observation that SB43542-treatment primarily reduced microenvironmental abnormalities suggests that the two drugs may have synergistic effects in the treatment of myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

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