• Molecules and Cells
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• 제27권3호
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• pp.359-363
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• 2009

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

• Parasites, Hosts and Diseases
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• 제58권6호
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• pp.675-679
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• 2020

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507-#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

• 약학회지
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• 제50권2호
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• pp.93-98
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• 2006

Human foamy virus (HFV) integrase mediates integration of viral c-DNA into cellular DNA. In this process, HFV prointegration complex (PIC) in which integrase is a key component moves to nuclei of the infected cells and leads to integration of viral DNA to the cellular genome, which is essential in viral life cycle. In general nuclear localization signals (NLS) have been suggested to be involved in localizing retroviral PIC to nuclei, but the mechanisms for nuclear localization of the HFV PIC remains unclear. To functionally identify the NLS of HFV integrase, various subdomains of the protein were expressed as GFP fusions and their subcellular locations were analyzed with confocal laser scanning microscopy. Wild type HFV integrase was karyophilic by targeting the fusion protein to nuclei of the COS-1 and 293T cells. Our results showed that strong NLS of HFV integrase was mapped to the C-terminal regions. In addition the karyophilic properties of N-terminal and central regions are not individually strong enough to direct localization of the fusion proteins to nuclei, but their cooperative activity for nuclear import was confirmed.

• Nuclear Engineering and Technology
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• 제54권1호
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• pp.193-206
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• 2022

Several algorithms for nuclear power plant (NPP) break event detection, isolation, localization, and size estimation are proposed. A break event can be promptly detected and isolated after its occurrence by simultaneously monitoring changes in the sensing readings and by employing an interquartile range-based isolation scheme. By considering the multi-sensor data block of a break to be rank-one, it can be located as the position whose lead field vector is most orthogonal to the noise subspace of that data block using the Multiple Signal Classification (MUSIC) algorithm. Owing to the flexibility of deep neural networks in selecting the best regression model for the available data, we can estimate the break size using multiple-sensor recordings of the break regardless of the sensor types. The efficacy of the proposed algorithms was evaluated using the data generated by Maanshan NPP simulator. The experimental results demonstrated that the MUSIC method could distinguish two near breaks. However, if the two breaks were close and of small sizes, the MUSIC method might wrongly locate them. The break sizes estimated by the proposed deep learning model were close to their actual values, but relative errors of more than 8% were seen while estimating small breaks' sizes.

• Molecules and Cells
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• 제37권2호
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• pp.140-148
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• 2014

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R ${\rightarrow}$ T), 313(R ${\rightarrow}$ T), 315(R ${\rightarrow}$ P), and 329(R ${\rightarrow}$ T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R ${\rightarrow}$ T), 318(K ${\rightarrow}$ T), and 324(K ${\rightarrow}$ T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.

• Biomolecules & Therapeutics
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• 제15권2호
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• pp.78-82
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• 2007

The proto-oncogene protein DEK is a nuclear binding phosphoprotein that has been associated with various human diseases including leukemia. Histone acetylation is an important post-translational modification which plays important role in transcriptional regulation. Auto-acetylation of histone acetyltransferase PCAF results in increment of its HAT activity and facilitation of its nuclear localization. In this study, we report that DEK inhibits PCAF auto-acetylation through direct interaction. The C-terminal acidic domains of DEK are responsible for the interaction with PCAF. Using confocal microscopy, we have shown that nuclear localization of PCAF is severely inhibited by DEK. Taken together, our results suggest that DEK may be involved in various cellular signal transduction pathways accommodated by PCAF through the regulation of PCAF auto-acetylation.

• Mycobiology
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• 제50권5호
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• pp.259-268
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• 2022

The nuclear import of proteins is a fundamental process in the eukaryotes including plant. It has become evident that such basic process is exploited by nuclear effectors that contain nuclear localization signal (NLS) and are secreted into host cells by fungal pathogens of plants. However, only a handful of nuclear effectors have been known and characterized to date. Here, we first summarize the types of NLSs and prediction tools available, and then delineate examples of fungal nuclear effectors and their roles in pathogenesis. Based on the knowledge on NLSs and what has been gleaned from the known nuclear effectors, we point out the gaps in our understanding of fungal nuclear effectors that need to be filled in the future researches.

• BMB Reports
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• 제47권6호
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• pp.342-347
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• 2014

Histone acetylation/deacetylation has been known to be associated with the transcriptional regulation of various genes. The role of histone deacetylase-3 in the expression regulation of MDR1 was investigated. The expression level of HDAC3 showed an inverse relationship with the expression level of MDR1. Wild-type HDAC3, but not catalytic mutant $HDAC3^{S424A}$, negatively regulated the expression of MDR1. Wild-type HDAC3, but not catalytic mutant $HDAC3^{S424A}$, showed binding to the promoter sequences of HDAC3. HDAC3 regulated the expression level, and the binding of Ac-$H3^{K9/14}$ and Ac-$H4^{K16}$ around the MDR1 promoter sequences. The nuclear localization signal domain of HDAC3 was necessary, and sufficient for the binding of HDAC3 to the MDR1 promoter sequences and for conferring sensitivity to microtubule-targeting drugs.

• Bulletin of the Korean Chemical Society
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• 제30권9호
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• pp.1981-1984
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• 2009

Subcellular localization of protein kinase often plays an important role in determining its activity and specificity. Protein kinase C (PKC), a family of multi-gene protein kinases has long been known to be translocated to the particular cellular compartments in response to DAG or its analog phorbol esters. We used C-terminal green fluorescent protein (GFP) fusion proteins of PKC isoforms to visualize the subcellular distribution of individual PKC isoforms. Intracellular localization of PKC-GFP proteins was monitored by fluorescence microscopy after transient transfection of PKC-GFP expression vectors in the HeLa cells. In unstimulated HeLa cells, all PKC isoforms were found to be distributed throughout the cytoplasm with a few exceptions. PKC$\theta$ was mostly localized to the Golgi, and PKC$\gamma$, PKC$\delta$ and PKC$\eta$ showed cytoplasmic distribution with Golgi localization. DAG analog TPA induced translocation of PKC-GFP to the plasma membrane. PKC$\alpha$, PKC$\eta$ and PKC$\theta$ were also localized to the Golgi in response to TPA. Only PKC$\delta$ was found to be associated with the nuclear membrane after transient TPA treatment. These results suggest that specific PKC isoforms are translocated to different intracellular sites and exhibit distinct biological effects.

• Animal cells and systems
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• 제11권1호
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• pp.39-50
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• 2007

Tau plays a role in numerous neuronal processes, such as vesicle transport, microtubule-plasma membrane interaction and intracellular localization of proteins. SUMO (Small Ubiquitin-like Modifier) modification (SUMOylation) appears to regulate diverse cellular processes including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin-dependent degradation, as well as gene transcription. We noticed that putative SUMOylation site is localized at $^{340}K$ of $Tau(^{339}VKSE^{342})$ with the consensus sequence information (${\Phi}KxE$ ; where ${\Phi}$ represents L, I, V or F and x is any amino acid). In this report, we demonstrated that $^{340}K$ of Tau is the SUMOylation site and that a point mutant of Tau S214E (an analog of the phospho $^{214}S$ Tau) promotes its SUMOylation at $^{340}K$ and its nuclear or nuclear vicinity localization, by co-immunoprecipitation and confocal microscopy analysis. Further, we demonstrate that the Tau S214E (neither Tau S214A nor Tau K340R) mutant increases its protein stability. However, the SUMOylation at $^{340}K$ of Tau did not influence cell survival, as determined by FACS analysis. Therefore, our results suggested that the phosphorylation of Tau on $^{214}S$ residue promotes its SUMOylation on $^{340}K$ residue and nuclear vicinity localization, and increases its stability, without influencing cell survival.