OBJECTIVE: To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study. METHODS: In the study, we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM. RESULTS: Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids, followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells. CONCLUSION GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.
Amino Acid Sequence ; Cytoplasm ; GTP Phosphohydrolases ; Humans ; Membrane Proteins ; Nuclear Export Signals ; Nuclear Localization Signals ; Recombinant Fusion Proteins ; Transfection

In order to investigate the mechanism of stat3 nuclear import. positioned a characterized NLS of the SV40 large T antigen into Stat3-GFP, Dstat3-GFP respectively between the C-terminus of Stat3 and the N-terminus of GFP to create Stat3-NLS-GFP and Dstat3-NLS-GFP. With NLS-GFP as the positive control, Expression of the Stat3-NLS-GFP without IL-6 stimulation and Stat3-GFP with IL-6 stimulation resulted in a predominantly nuclear localization in 293T cell. Expression of Stat3-GFP and Dstat3-NLS-GFP without IL-6 stimulation resulted in predominantly cytoplasm localization in 293T cell. The results suggest that latent Stat3 is not anchored in the cytoplasm, and that nuclear localization in response to IL-6 is facilitated by gain of an NLS function.
Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Nuclear Localization Signals ; Plasmids ; metabolism ; STAT3 Transcription Factor ; metabolism

Nuclear targeting of bacterial proteins in host cells and subsequent interaction with nuclear molecules are an emerging pathogenic mechanism of bacteria. In this study, we predicted the nuclear targeting proteins with nuclear localization signals (NLSs) in Staphylococcus aureus using bioinformatic analysis. A total of 51 proteins of S. aureus, comprising of 24 functional and 27 hypothetical proteins, were predicted to carry putative NLSs. Among them, beta-lactamase and MsrR proteins with the putative NLSs were selected to determine the nuclear targeting in host cells. Fusion proteins of BlaZ-green fluorescent protein (GFP) were evenly distributed in the nuclei of host cells and subsequently induced host cell death. However, fusion proteins of MsrR-GFP were not localized in the nuclei of host cells In conclusion, screening of nuclear targeting proteins with NLSs and determination of their pathology in host cells may open up the new field of S. aureus pathogenesis.
Bacteria ; Bacterial Proteins ; beta-Lactamases* ; Cell Death ; Computational Biology ; Mass Screening ; Nuclear Localization Signals* ; Pathology ; Staphylococcus aureus*

Transportation between the cytoplasm and the nucleoplasm is critical for many physiological and pathophysiological processes including gene expression, signal transduction, and oncogenesis. So, the molecular mechanism for the transportation needs to be studied not only to understand cell physiological processes but also to develop new diagnostic and therapeutic targets. Recent progress in the research of the nuclear transportation (import and export) via nuclear pore complex and four important factors affecting nuclear transport (nucleoporins, Ran, karyopherins, and nuclear localization signals/nuclear export signals) will be discussed. Moreover, the clinical significance of nuclear transport and its application will be reviewed. This review will provide some critical insight for the molecular design of therapeutics which need to be targeted inside the nucleus.
Active Transport, Cell Nucleus* ; Carcinogenesis ; Cell Physiological Processes ; Cytoplasm ; Gene Expression ; Karyopherins ; Nuclear Localization Signals ; Nuclear Pore ; Nuclear Pore Complex Proteins ; Signal Transduction ; Transportation

The Cap gene of antisense strand of circovirus has the most variation of the genome, and encodes a capsid protein which has the main immunogenicity. The N-terminal of capsid protein makes up of nuclear localization signal which is involved with virus location. This review summarizes the research advance of Cap gene of circovirus in the sequence characteristics, its encoding capsid protein, basic functions of the capsid protein and its interaction with MKRN1 protein, Hsp40 protein, receptor protein gClqR and complement factor C1qB protein. This paper lays a theory foundation for the further study of the capsid protein in the aspects of viral attachment, replication and transportation.
Animals ; Capsid Proteins ; genetics ; immunology ; metabolism ; Circoviridae Infections ; veterinary ; virology ; Circovirus ; genetics ; immunology ; Genetic Variation ; Genome, Viral ; genetics ; Nuclear Localization Signals ; Protein Binding ; Virus Replication

To investigate the effect of the localization of oligonucleotides decoy (ODNs decoy) on the activation of nuclear factor-kappaB (NF-kappaB) in TNF-alpha induced HeLa cells. The mercapto group-modified nuclear localization signal (NLS) peptide was covalently conjugated to amino group-modified NF-kappaB ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent. The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope. The cell viability was detected by MTT assay, and then the activity of NF-kappaB in cell nuclear extract was assayed by electrophoretic mobility shift assay (EMSA). The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%. It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy, while NLS-ODNs decoy significantly inhibited the activation of NF-kappaB in TNF-alpha induced HeLa cells nuclear extracts. This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus, and thus improve its inhibitory effects on the activation a transcription factor.
Base Sequence ; Cell Nucleus ; metabolism ; HeLa Cells ; Humans ; Molecular Sequence Data ; NF-kappa B ; genetics ; metabolism ; Nuclear Localization Signals ; genetics ; Oligonucleotides ; genetics ; metabolism ; Transfection

Abstract:By using PVX derived vector pGR107, the effect of BYDV-MP nuclear localization signal on the movement of PVX was studied. BYDV-MP was cloned into pGR107 using GFP as an indicator. BYDV-MP was then shown to induce the systemic infection and exacerbate the symptom of PVX through infecting Nicotiana benthamiana. When the PVX gene encoding 25kD protein, which functioned as a systematic movemnet protein,was deleted and the above experiment was repeated, the result showed that BYDV-MP could compensate the systemic movement of PVX. A serial mutants with substitutions on the fifth, sixth and seventh amino acids of BYDV-MP nuclear localization signal was further constructed. It was found that the mutants at the fifth, sixth amino acids in BYDV-MP nuclear localization signal could only delay or weaken systemic movement of PVX whereas the mutant at seventh amino acid could entirely inhibit systemic movement of PVX.
Amino Acid Sequence ; Green Fluorescent Proteins ; genetics ; Luteovirus ; physiology ; Molecular Sequence Data ; Nuclear Localization Signals ; chemistry ; physiology ; Plant Viral Movement Proteins ; physiology ; Potexvirus ; genetics ; physiology

OBJECTIVETo establish a new method for studying the mechanism of nuclear localization signal (NLS)-mediated nuclear translocation in living cells.

METHODSThe cells were treated with 67 mg/L 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), followed by incubation with 1 g/L wheat germ agglutinin (WGA), and their effects on interferon- γ (IFN-γ)-induced nuclear translocation of signal transducer and activator of transcription 1 (STAT1) were observed.

RESULTSTreatment with CHAPS alone had no effect on IFN-γ-induced nuclear translocation of STAT1, while this process was blocked by further WGA incubation.

CONCLUSIONWe established a new, simple but effective method for studying the mechanism of NLS-mediated nuclear translocation in living cells by perforating the cell membrane with CHAPS treatment.

Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Cholic Acids ; Cytological Techniques ; HeLa Cells ; Humans ; Interferon-gamma ; metabolism ; Nuclear Localization Signals ; metabolism ; STAT1 Transcription Factor ; metabolism ; Signal Transduction

The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin beta. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na+/H+ exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin beta, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a 'pH-dependent' NLS.
Active Transport, Cell Nucleus ; Amino Acid Sequence ; HeLa Cells ; Humans ; Hydrogen-Ion Concentration ; Molecular Sequence Data ; *Nuclear Localization Signals ; Protein Structure, Tertiary ; Proto-Oncogene Proteins c-met/*analysis/genetics/*metabolism ; Sequence Deletion

The recovery of the virus from genetic materials in in vitro culture systems or sensitive animals is called virus rescue. A functional infectious clone of RNA virus provides unlimited possibility for genetic studies and the related reverse genetics system that allows directed genetic manipulation of an RNA virus is an extremely powerful research tool. In the past twenty years, especially since the first infectious clone of a negative-stranded RNA virus was reported in the mid-1990's, the reverse genetics systems have been available for nearly all the major human and animal RNA virus groups. The article reviews the progress of this technology, highlighting the obstacles in the construction of reverse genetics systems for major groups of human as well as animal RNA viruses and how the virologists overcame them. There are mainly four external expression systems for construction of the RNA virus reverse genetics systems basing on the kind of RNA viruses. These systems include in vitro RNA transcripts, RNA polymerase I-driven expression plasmids, RNA polymerase II-driven expression plasmids and modified vaccinia virus/T7 RNA polymerase-driven expression system. In particular, the viral nucleoprotein and polymerase proteins are required to assemble the viral ribonucleoprotein (RNP) complexes for the rescue of the negative-stranded RNA viruses. Relevant topics about the rescue of the typical viruses are discussed, including poliovirus with the de novo synthesis, Coronaviridae with the largest size of genome, Flaviviridae with the instable clones, HCV with the quasispecies nature, nodaviruses with the virus-host interaction, influenza virus with the RNA pol I transcription system, Arenavirdae with the ambisense coding strategies etc.
Animals ; DNA, Complementary ; genetics ; Genetic Engineering ; Humans ; Nuclear Localization Signals ; genetics ; RNA Viruses ; genetics ; isolation & purification ; pathogenicity ; RNA, Viral ; biosynthesis ; genetics ; Ribonucleoproteins ; biosynthesis ; metabolism ; Transcription, Genetic ; Transfection ; Virus Replication