Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1

ABSTRACT Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In Schizosaccharomyces pombe, the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys13, Arg16, Arg19, Lys24, Arg28, Lys45, Arg47, Arg50, and Arg53 residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif, 328Cys-X-Cys-X3-Cys-X-Cys-X2-Cys-X2-His342 (designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in trans and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.

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Identification and characterization of a nucleolar phosphoprotein, Nopp140, as a transcription factor.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.230 ◽

1997 ◽

Vol 17 (1) ◽

pp. 230-239 ◽

Cited By ~ 46

Author(s):

L H Miau ◽

C J Chang ◽

W H Tsai ◽

S C Lee

Keyword(s):

Transcription Factor ◽

Gene Activation ◽

Binding Motif ◽

Physical Interaction ◽

Dependent Manner ◽

Positive Regulation ◽

Enhancer Binding Protein ◽

Simplex Virus

Expression of the alpha-1 acid glycoprotein (AGP) gene (agp) is activated by a key transcription factor, AGP/enhancer-binding protein (AGP/EBP, commonly called C/EBP beta), in the liver during the acute-phase response. In addition to this positive regulation, agp is negatively regulated by nucleolin (T. H. Yang et al., Mol. Cell. Biol. 14:6068-6074, 1994). Other factors involve in positive regulation of the agp gene are poorly characterized. In a systematic search for factors that may interact with AGP/EBP, we have identified Nopp 140, a phosphoprotein of 140 kDa, by immunoaffinity chromatography. Nopp 140 not only functions as a transcriptional activator per se but also interacts with AGP/EBP to synergistically activate the agp gene in an AGP/EBP-binding motif-dependent manner. In addition to interacting with AGP/EBP, Nopp140 interacts specifically with TFIIB. Distinct regions of Nopp140 that interact with AGP/EBP and TFIIB have been characterized. The sequence of Nopp140 contains several stretches of serine- and acidic amino acid-rich sequences which are also found in ICP4 of herpes simplex virus type 1, a known transcription factor that interacts with TFIIB. The physical interaction between TFIIB and wild-type Nopp140 or several deletion mutants of Nopp140 correlates with the ability of Nopp140 to activate the agp gene synergistically with AGP/EBP. Thus, the molecular mechanism for agp gene activation may involve the interaction of AGP/EBP and TFIIB mediated by coactivator Nopp140.

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Involvement of the ubiquitous Oct-1 transcription factor in hormonal induction of β-casein gene expression

Biochemical Journal ◽

10.1042/bj20060570 ◽

2006 ◽

Vol 401 (1) ◽

pp. 57-64 ◽

Cited By ~ 20

Author(s):

Bing Dong ◽

Feng-Qi Zhao

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Binding Site ◽

Promoter Activity ◽

Regulatory Region ◽

Binding Motif ◽

Dependent Manner ◽

Casein Gene ◽

Hormonal Induction ◽

Lactogenic Hormones

Transcription of the milk protein β-casein gene is induced by the lactogenic hormones Prl (prolactin) and glucocorticoids. Multiple transcription factors involved in this induction have been identified, including the STAT5 (signal transducer and activator of transcription 5) and the GR (glucocorticoid receptor). Our previous studies have identified a binding site for the ubiquitous Oct-1 (octamer-binding transcription factor 1) protein in the lactogenic hormonal regulatory region of the mouse β-casein promoter. In the present study, we report that Oct-1 is indeed expressed and binds to the β-casein promoter in mammary epithelial cells. Oct-1 activates hormonally induced β-casein promoter activity in a dose-dependent manner. Hormonal induction of promoter activity was decreased not only by mutating the Oct-1-binding site from ATTAGCAT to GCTAGCAT, which abolishes Oct-1 binding (50% decrease, P<0.01), but also by changing the site to the consensus Oct-1-binding motif ATTTGCAT (40% decrease, P<0.01). Reversing the Oct-1-binding site reduced hormonal induction by 70% (P<0.01), showing that orientation of Oct-1 binding is also critical in hormonal action. In transient transfection experiments, Oct-1 collaboratively transactivated the β-casein gene promoter with STAT5 and/or GR in the presence of Prl receptor in cells treated with the lactogenic hormones. The C-terminus of Oct-1 was not essential to its function. The results of the present study provide biochemical evidence that the ubiquitous Oct-1 transcription factor may be involved in hormonally regulated, tissue-specific β-casein gene expression.

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Faculty Opinions recommendation of Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1142881.599970 ◽

2009 ◽

Author(s):

Daniel Bikle

Keyword(s):

Nuclear Localization ◽

Growth Retardation ◽

Nuclear Localization Sequence ◽

Related Protein ◽

C Terminus ◽

Localization Sequence ◽

Severe Growth ◽

Early Lethality

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Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.2.c248 ◽

2001 ◽

Vol 280 (2) ◽

pp. C248-C253 ◽

Cited By ~ 90

Author(s):

Stephen C. Dahl ◽

Joseph S. Handler ◽

H. Moo Kwon

Keyword(s):

Transcription Factor ◽

Nuclear Localization ◽

Kinase Inhibitors ◽

Dependent Manner ◽

Binding Assays ◽

P38 Inhibitor ◽

Vitro Binding ◽

Compatible Osmolytes

The accumulation of compatible osmolytes during osmotic stress is observed in virtually all organisms. In mammals, the hypertonicity-induced expression of osmolyte transporters and synthetic enzymes is conferred by the presence of upstream tonicity-responsive enhancer (TonE) sequences. Recently, we described the cloning and initial characterization of TonE-binding protein (TonEBP), a transcription factor that translocates to the nucleus and associates with TonE sequences in a tonicity-dependent manner. We now report that hypertonicity induces an increase in TonEBP phosphorylation that temporally correlates with increased nuclear localization of the molecule. TonEBP phosphorylation is not affected by a number of kinase inhibitors, including the p38 inhibitor SB-203580. In addition, in vitro binding assays show that the association of TonEBP with TonE sequences is not affected by phosphorylation. Thus TonEBP phosphorylation is an early step in the response of cells to hypertonicity and may be required for nuclear import or retention.

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IPSE, a Parasite-Derived, Host Immunomodulatory Infiltrin Protein, Alleviates Resiniferatoxin-Induced Bladder Pain

10.1101/2020.06.11.146829 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kenji Ishida ◽

Evaristus C. Mbanefo ◽

Loc Le ◽

Olivia Lamanna ◽

Luke F. Pennington ◽

...

Keyword(s):

Nuclear Localization ◽

Analgesic Effect ◽

Transient Receptor Potential ◽

Nuclear Localization Sequence ◽

Therapeutic Effects ◽

Dependent Manner ◽

Bladder Pain ◽

Nociceptive Neurons ◽

Sequence Dependent ◽

Localization Sequence

AbstractThe transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is an important mediator of nociception and its expression is enriched in nociceptive neurons. TRPV1 signaling has been implicated in bladder pain and is a potential analgesic target. Resiniferatoxin is the most potent known agonist of TRPV1. Acute exposure of the rat bladder to resiniferatoxin has been demonstrated to result in pain-related freezing and licking behaviors that are alleviated by virally encoded IL-4. The interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE) is a powerful inducer of IL-4 secretion, and is also known to alter host cell transcription through a nuclear localization sequence-dependent mechanism. We previously reported that IPSE ameliorates ifosfamide-induced bladder pain in an IL-4- and nuclear localization sequence-dependent manner. We hypothesized that pre-administration of IPSE to resiniferatoxin-challenged mice would dampen pain-related behaviors. IPSE indeed lessened resiniferatoxin-triggered freezing behaviors in mice. This was a nuclear localization sequence-dependent phenomenon, since administration of a nuclear localization sequence mutant version of IPSE abrogated IPSE’s analgesic effect. In contrast, IPSE’s analgesic effect did not seem IL-4-dependent, since use of anti-IL-4 antibody in mice given both IPSE and resiniferatoxin did not dramatically affect freezing behaviors. RNA-Seq analysis of resiniferatoxin- and IPSE-exposed bladders revealed differential expression of TNF/NF-κb-related signaling pathway genes. In vitro testing of IPSE uptake by urothelial cells and TRPV1-expressing neuronal cells showed uptake by both cell types. Thus, IPSE’s nuclear localization sequence-dependent therapeutic effects on TRPV1-mediated bladder pain may act on TRPV1-expressing neurons and/or may rely upon urothelial mechanisms.

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Cytoplasmic P53 Immunostaining With N-Terminus P53 Antibody And Absence Of Staining With C-Terminus P53 Antibody: A Report Of Two Pediatric Sarcomas With Distal Truncating TP53 Mutations Affecting Nuclear Localization Domain

International Journal of Surgical Pathology ◽

10.1177/10668969211065115 ◽

2021 ◽

pp. 106689692110651

Author(s):

Hilda Mirbaha ◽

Deyssy Carrillo ◽

Midori Mitui ◽

Matthew C. Hiemenz ◽

Vivekanand Singh ◽

...

Keyword(s):

Nuclear Localization ◽

Immunohistochemical Staining ◽

Precise Description ◽

Cytoplasmic Staining ◽

Tp53 Mutations ◽

Localization Domain ◽

C Terminus ◽

Pediatric Sarcomas ◽

N Terminus ◽

P53 Antibody

P53 immunohistochemical staining with antibodies targeted to epitopes at or near the N-terminus are commonly used in diagnostic pathology practice as a surrogate for TP53 mutations. The abnormal staining patterns indicating TP53 mutations include nuclear overexpression, null, and the recently described cytoplasmic staining. The latter staining pattern occurs with the less common TP53 mutations affecting its nuclear localization and/or tetramerization domains that are located toward the C-terminus. Here we describe the first two cases of pediatric sarcomas with cytoplasmic staining with P53 antibody against N-terminus epitope and the absence of staining with P53 antibody against C-terminus epitope. We propose that a more precise description of P53 immunohistochemical staining patterns should include the nature of the antibody used.

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Regulation of mRNA export through API5 and nuclear FGF2 interaction

Nucleic Acids Research ◽

10.1093/nar/gkaa335 ◽

2020 ◽

Vol 48 (11) ◽

pp. 6340-6352 ◽

Cited By ~ 3

Author(s):

Seoung Min Bong ◽

Seung-Hyun Bae ◽

Bomin Song ◽

HyeRan Gwak ◽

Seung-Won Yang ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Export ◽

Mrna Export ◽

Structural Basis ◽

Nuclear Localization Sequence ◽

Physical Interaction ◽

Dynamic Regulation ◽

Localization Sequence ◽

Localization Signal ◽

Apoptosis Inhibitor

Abstract API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5–FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.

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Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

Journal of Bacteriology ◽

10.1128/jb.00135-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4001-4016 ◽

Cited By ~ 26

Author(s):

Wallace A. Kaserer ◽

Xiaoxu Jiang ◽

Qiaobin Xiao ◽

Daniel C. Scott ◽

Matthew Bauler ◽

...

Keyword(s):

Amino Acids ◽

Outer Membrane ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

Binding Motif ◽

Inner Membrane ◽

E Coli ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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