Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells

A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.

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Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 Vpr

Journal of Virology ◽

10.1128/jvi.75.3.1522-1532.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1522-1532 ◽

Cited By ~ 85

Author(s):

Michael P. Sherman ◽

Carlos M. C. de Noronha ◽

Marina I. Heusch ◽

Spencer Greene ◽

Warner C. Greene

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Import ◽

Nuclear Export ◽

Fusion Proteins ◽

Nucleocytoplasmic Shuttling ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin α/β-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1–71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK–Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich α-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

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Impaired telomerase activity in human cells expressing GFP-Ku86 fusion proteins

Cytogenetic and Genome Research ◽

10.1159/000167819 ◽

2008 ◽

Vol 122 (3-4) ◽

pp. 326-335 ◽

Cited By ~ 1

Author(s):

C. Badie ◽

R.J. Yáñez-Muñoz ◽

C. Muller ◽

B. Salles ◽

A.C.G. Porter

Keyword(s):

Fusion Proteins ◽

Telomerase Activity ◽

Human Cells

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Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis

The Plant Journal ◽

10.1046/j.1365-313x.2000.00923.x ◽

2000 ◽

Vol 24 (6) ◽

pp. 837-847 ◽

Cited By ~ 143

Author(s):

Liqun Du ◽

Zhixiang Chen

Keyword(s):

Salicylic Acid ◽

Dna Binding ◽

Protein Kinases ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Genes Encoding

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A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm ofEscherichia coli

Protein Expression and Purification ◽

10.1016/s1046-5928(05)80026-9 ◽

1992 ◽

Vol 3 (2) ◽

pp. 108-113

Author(s):

H ENGEL ◽

H MOTTL ◽

W KECK

Keyword(s):

Fusion Proteins ◽

Protein A ◽

Ofescherichia Coli ◽

Staphylococcal Protein A ◽

Staphylococcal Protein ◽

High Level

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Alpha-Tocopherol Protects Cultured Human Cells from the Acute Lethal Cytotoxicity of Dioxin

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831.72.3.147 ◽

2002 ◽

Vol 72 (3) ◽

pp. 147-153 ◽

Cited By ~ 8

Author(s):

Kei-Ichi Hirai ◽

Jie-Hong Pan ◽

Ying-Bo Shui ◽

Eriko Simamura ◽

Hiroki Shimada ◽

...

Keyword(s):

Cell Death ◽

Cell Damage ◽

Smooth Endoplasmic Reticulum ◽

Dehydroascorbic Acid ◽

Human Cells ◽

Alpha Tocopherol ◽

Cervical Cancer Cells ◽

Cultured Human Cells ◽

Nuclear Damage ◽

Conjunctival Epithelial Cells

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30–40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0–31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78–97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.

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