Creation of a protein vector construct including an SSBTne DNA-binding domain and VirD2 nuclear localization signal

2008 ◽  
Vol 34 (5) ◽  
pp. 329-331
Author(s):  
D. V. Grishin ◽  
V. P. Gudov ◽  
O. V. Sergienko ◽  
V. G. Lunin ◽  
P. N. Kharchenko
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

2012 ◽  
Vol 11 (12) ◽  
pp. 1441-1450 ◽  
Author(s):  
Hong-Ming Hsu ◽  
Yu Lee ◽  
Dharmu Indra ◽  
Shu-Yi Wei ◽  
Hsing-Wei Liu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Trichomonas Vaginalis ◽  
Structural Integrity ◽  
Nuclear Translocation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Nuclear Localization Signals ◽  
Localization Signal

ABSTRACTInTrichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631 ◽  
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Metal-Responsive Transcription Factor 1 (MTF-1) Activity Is Regulated by a Nonconventional Nuclear Localization Signal and a Metal-Responsive Transactivation Domain

2009 ◽  
Vol 29 (23) ◽  
pp. 6283-6293 ◽  
Author(s):  
Uschi Lindert ◽  
Mirjam Cramer ◽  
Michael Meuli ◽  
Oleg Georgiev ◽  
Walter Schaffner
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Localization Signal ◽  
Transcription Factor 1

ABSTRACT Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; “humanizing” mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

scholarly journals Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B.

1996 ◽  
Vol 16 (10) ◽  
pp. 5444-5449 ◽  
Author(s):  
H Suyang ◽  
R Phillips ◽  
I Douglas ◽  
S Ghosh
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Stable Complex ◽  
Dna Binding Domain ◽  
Nf Kappa B ◽  
Prolonged Stimulation ◽  
I Kappa B Alpha ◽  
Localization Signal

Stimulation with inducers that cause persistent activation of NF-kappa B results in the degradation of the NF-kappa B inhibitors, I kappa B alpha and I kappa B beta. Despite the rapid resynthesis and accumulation of I kappa B alpha, NF-kappa B remains induced under these conditions. We now report that I kappa B beta is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated I kappa B beta forms a stable complex with NF-kappa B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-kappa B, and the I kappa B beta-NF-kappa B complex enters the nucleus. It appears therefore that during prolonged stimulation, I kappa B beta functions as a chaperone for NF-kappa B by protecting it from I kappa B alpha and allowing it to be transported to the nucleus.

scholarly journals Simian Virus 40 Vp1 DNA-Binding Domain Is Functionally Separable from the Overlapping Nuclear Localization Signal and Is Required for Effective Virion Formation and Full Viability

2001 ◽  
Vol 75 (16) ◽  
pp. 7321-7329 ◽  
Author(s):  
Peggy P. Li ◽  
Akira Nakanishi ◽  
Dorothy Shum ◽  
Peter C.-K. Sun ◽  
Adler M. Salazar ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Carboxy Terminal

ABSTRACT A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1ΔC58 pentamer bound DNA with aKd of 1.8 × 10−9 M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1ΔC17 had comparable apparentKd s of 5.3 × 10−9 to 7.3 × 10−9 M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1ΔC58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 · 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1ΔC58 level for NLSm1 · 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209–8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1 · 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 · 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.

scholarly journals A novel nuclear localization signal spans the linker of the two DNA-binding subdomains in the conserved paired domain of Pax6

2018 ◽  
Vol 93 (2) ◽  
pp. 75-81 ◽  
Author(s):  
Hiromasa Tabata ◽  
Akihiro Koinui ◽  
Atsushi Ogura ◽  
Daisuke Nishihara ◽  
Hiroaki Yamamoto
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Paired Domain ◽  
Localization Signal

scholarly journals Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Richard Izrael ◽  
Lívia Marton ◽  
Gergely N. Nagy ◽  
Hajnalka L. Pálinkás ◽  
Nóra Kucsma ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Line ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Membrane Binding ◽  
Binding Domain ◽  
Regulatory Function ◽  
Phosphocholine Cytidylyltransferase ◽  
Phosphatidylcholine Biosynthesis ◽  
Localization Signal

AbstractThe phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

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