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1

Quantification of progesterone binding in mammary tissue of pregnant ewes  

SciTech Connect

Progestin-binding sites in mammary tissue from 14 prepartum, multiparous ewes at 50, 80, 115, and 140 d of gestation were demonstrated by the binding of (/sup 3/H) R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) to ovine mammary cytosol in the presence of sodium molybdate and excess cortisol. Homogenization extracted 89% of total mammary receptors (nuclear) into cytosol. Binding was specific for progestins and was of high affinity. The average dissociation constant for (/sup 3/H) R5020 specifically bound to receptors extracted into mammary cytosol was 1.9 (+/- .4) x 10/sup -9/ M (n = 14) and did not change significantly over the test period. However, binding capacities (fmol/mg cytosolic protein) differed according to stage of gestation with averages of 125 +/- 53, 149 +/- 26, 656 +/- 216, 57 +/- 22 at 50, 80, 115, and 140 d of pregnancy, respectively. Increased number of progestin-binding sites at 115 d of gestation (whether data are expressed per unit of tissue weight, DNA, or cytosolic protein) suggests that an increase per mammary epithelial cell may be necessary to produce the full lobuloalveolar proliferation observed at this stage of gestation.

Smith, J.J.; Capuco, A.V.; Akers, R.M.

1987-06-01

2

A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone  

E-print Network

on the perineum (1). This model system underscores the behavior's dependence on E, P, and tactile stimulation (2 of the neural circuitry responsible for lordosis. One brain structure important for integrating the endocrine.g., Hsc73) (5) and others that may serve specific functions in intracellular trafficking in endocrine

Jarvis, Erich D.

3

Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.  

PubMed

Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels. PMID:16901474

Albani, J R

2006-11-01

4

Emerging roles of nuclear protein phosphatases  

Microsoft Academic Search

The phosphorylation state of any protein represents a balance of the actions of specific protein kinases and protein phosphatases. Many protein phosphatases are highly enriched in, or exclusive to, the nuclear compartment, where they dephosphorylate key substrates to regulate various nuclear processes. In this review we will discuss recent findings that define the role of nuclear protein phosphatases in controlling

Laura Trinkle-Mulcahy; Annegret Ulke-Lemée; Greg B. G. Moorhead

2007-01-01

5

Nuclear matrix proteins and hereditary diseases  

Microsoft Academic Search

The review summarizes literature data on alterations of structure or expression of different nuclear matrix proteins in hereditary syndromes. From the point of view of involvement of nuclear matrix proteins in etiology and pathogenesis of the disease, hereditary pathologies can be classified in pathologies with pathogenesis associated with defects of nuclear matrix proteins and pathologies associated to changes of the

N. Sjakste; T. Sjakste

2005-01-01

6

Nuclear localization and the heat shock proteins  

Microsoft Academic Search

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs\\u000a are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization\\u000a of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization\\u000a occurring in response

A. A. Knowlton; M. Salfity

1996-01-01

7

GAPDH Mediates Nitrosylation of Nuclear Proteins  

PubMed Central

S-nitrosylation by nitric oxide (NO) is a major mode of signaling to cellular proteins1, including prominent nuclear proteins such as HDAC22 and PARP13. The high reactivity of the NO group with protein thiols implies the existence of selective targeting mechanisms. Specificity of NO signaling is often achieved by the binding of NO synthase (NOS) to target proteins, either directly4 or through scaffolding proteins such as PSD-955 and CAPON6. As the three principal isoforms of NOS - neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) - are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys150 residue, conferring upon it the ability to bind to Siah1, which possesses a nuclear localization signal and conveys nitrosylated GAPDH (SNO-GAPDH) to the nucleus7. We now show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme SIRT1, histone deacetylase-2 (HDAC2), and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of NO groups may be a general mechanism in cellular signal transduction. PMID:20972425

Kornberg, Michael D.; Sen, Nilkantha; Hara, Makoto R.; Juluri, Krishna R.; Van K. Nguyen, Judy; Snowman, Adele M.; Law, Lindsey; Hester, Lynda D.; Snyder, Solomon H.

2010-01-01

8

Nuclear matrix proteins and hereditary diseases.  

PubMed

The review summarizes literature data on alterations of structure or expression of different nuclear matrix proteins in hereditary syndromes. From the point of view of involvement of nuclear matrix proteins in etiology and pathogenesis of the disease hereditary pathologies can be classified in pathologies with pathogenesis associated with defects of nuclear matrix proteins and pathologies associated to changes of the nuclear matrix protein spectrum. The first group includes laminopathies, hereditary diseases with abnormal nuclear-matrix associated proteins and triplet extension diseases associated with accumulation of abnormal proteins in the nuclear matrix. Laminopathies are hereditary diseases coupled to structural defects of the nuclear lamina. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Most of them are due to mutations in the lamin A/C gene, one - to mutations in emerin gene, some are associated with mutations in Lamin B receptor gene. In Werner's, Bloom's, Cockayne's syndromes, Fanconi anemia, multiple carboxylase deficiency mutations in nuclear matrix protein or enzyme gene lead to deficient DNA repair, abnormal regulation of cell growth and differentiation or other specific metabolic functions. Proteins with a long polyglutamic tract synthesized in the cells of patients with dentato-rubral and pallido-luysian atrophy, myotonic dystrophy and Huntington disease interfere with transcription on the nuclear matrix. Down's syndrome is a representative of the group of diseases with altered nuclear matrix protein spectrum. PMID:15865282

Sjakste, N; Sjakste, T

2005-03-01

9

Dynamic Nuclear Polarization of Deuterated Proteins  

E-print Network

For D's a jolly good fellow: Deuteration of proteins significantly increases the signal enhancements observed in dynamic nuclear polarization (DNP) magic-angle spinning (MAS) NMR experiments. In 13C?CP-MAS spectra an ...

Akbey, Umit

10

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins  

Microsoft Academic Search

An in vitro system was developed that pro- vides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation

Donald D. Newmeyer; Deborah R. Finlay; Douglass J. Forbes

1986-01-01

11

A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast.  

PubMed Central

We have isolated mutants of the yeast Saccharomyces cerevisiae that are defective in localization of nuclear proteins. Chimeric proteins containing the nuclear localization sequence from SV40 large T-antigen fused to the N-terminus of the mitochondrial F1 beta-ATPase are localized to the nucleus. Npl (nuclear protein localization) mutants were isolated by their ability to grow on glycerol as a consequence of no longer exclusively targeting SV40-F1 beta-ATPase to the nucleus. All mutants with defects in localization of nucleolar proteins and histones are temperature sensitive for growth at 36 degrees C. Seven alleles of NPL3 and single alleles of several additional genes were isolated. NPL3 mutants were studied in detail. NPL3 encodes a nuclear protein with an RNA recognition motif and similarities to a family of proteins involved in RNA metabolism. Our genetic analysis indicates that NPL3 is essential for normal cell growth; cells lacking NPL3 are temperature sensitive for growth but do not exhibit a defect in localization of nuclear proteins. Taken together, these results indicate that the mutant forms of Npl3 protein isolated by this procedure are interfering with nuclear protein uptake in a general manner. Images PMID:1392078

Bossie, M A; DeHoratius, C; Barcelo, G; Silver, P

1992-01-01

12

Identification of specific binding proteins for a nuclear location sequence  

Microsoft Academic Search

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm1. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex2,3 and are directed by specific nuclear location sequences (NLS) in the proteins4. NLS mediate the nuclear import of isolated nuclear proteins after microinjection

Stephen A. Adam; Thomas J. Lobl; Mark A. Mitchell; Larry Gerace

1989-01-01

13

Demystifying the nuclear function of Argonaute proteins.  

PubMed

The Argonaute family of proteins is highly evolutionarily conserved and plays essential roles in small RNA-mediated gene regulatory pathways and in a wide variety of cellular processes. They were initially discovered by genetics studies in plants and have been well characterized as key components of gene silencing pathways guided by small RNAs, a phenomenon known as RNA interference. Conventionally, guided by different classes of small RNAs, Argonautes bind to and silence homologous target sequences at the post-transcriptional level. Increasing lines of evidence support their multi-functional roles in the nucleus. Advances in high-throughput genome-wide methodologies have greatly facilitated our understanding of their functions in post-transcriptional gene silencing as well as in other nuclear events. In this point-of-view, we will summarize key findings from genome-wide analyses of the Ago subfamily of proteins in mammals and Drosophila, discuss their nuclear functions in the regulation of transcription and alternative splicing identified in recent years, and briefly touch upon their potential implications in cancer. PMID:24384674

Huang, Vera; Li, Long-Cheng

2014-01-01

14

Identification of specific binding proteins for a nuclear location sequence.  

PubMed

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus. PMID:2911368

Adam, S A; Lobl, T J; Mitchell, M A; Gerace, L

1989-01-19

15

Nuclear Protein Isoforms: Implications for Cancer Diagnosis and Therapy  

PubMed Central

Post translational modifications (PTMs) of nuclear proteins play essential roles in the regulation of gene transcription and signal transduction pathways. Numerous studies have demonstrated a correlation between specific nuclear protein isoforms and cellular malignant process. This communication reviews the impact of major PTM events such as phosphorylation, acetylation, methylation, ubiquitination and sumoylation on several important nuclear proteins including p53, histones, proliferating cellular nuclear antigen (PCNA), and retinoblastoma protein (Rb) in the process. In addition, the implications of the PTMs as cancer biomarkers and therapeutic targets are considered. PMID:21328449

Shen, Fei; Kirmani, Kashif Z.; Xiao, Zhimin; Thirlby, Benjamin H.; Hickey, Robert J.; Malkas, Linda H.

2011-01-01

16

Automated local bright feature image analysis of nuclear protein distribution identifies  

E-print Network

arrest proliferation, undergo apoptosis, or differentiate, distribution of nuclear proteins changes proliferation and differentiation (1, 2). Several nuclear proteins have been reported to display a specificAutomated local bright feature image analysis of nuclear protein distribution identifies changes

Knowles, David William

17

Nuclear DNA and nuclear protein content of tumor cell in adenocarcinoma of the lung.  

PubMed

We measured DNA and protein contents of nuclei in resected tumors from 17 cases (six female and 11 male) of pulmonary primary adenocarcinoma. The relationship between the contents of nuclear DNA and protein on tumoral behavior was evaluated. We found that the content of nuclear DNA and protein was significantly higher in advanced stages than in early stages in patients with adenocarcinoma (P < 0.05). We also found the nuclear protein, DNA contents and the ratio of nuclear protein to DNA significantly higher in patients under the average age of 61 than in the patients over 61 (P < 0.05). Tumor size was found to be greater in Stage III and IV cases than Stage I cases (P < 0.05). In conclusion, it has been postulated that evaluation of malignant disease and its behavior might be simplified by measuring nuclear DNA and the protein contents of tumors, contributing to disease control. PMID:8528636

Gönüllü, U; Kato, H

1995-08-01

18

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants  

SciTech Connect

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)] [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany)] [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)] [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

2009-08-15

19

GTP-binding proteins in rat liver nuclear envelopes  

SciTech Connect

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of (alpha-32P)GTP binding to Western-blotted proteins and UV crosslinking of (alpha-32P)GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and (alpha-32P)GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with (alpha-32P)GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all (alpha-32P)GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with (alpha-32P)ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membranes.

Rubins, J.B.; Benditt, J.O.; Dickey, B.F.; Riedel, N. (Boston Univ. School of Medicine, MA (USA))

1990-09-01

20

TTRAP is a novel PML nuclear bodies-associated protein  

SciTech Connect

PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-{gamma}, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.

Xu Guanlan; Pan Yukun; Wang Bingyin; Huang Lu; Tian Ling; Xue Jinglun [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai (China); Chen Jinzhong [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai (China)], E-mail: kingbellchen@fudan.edu.cn; Jia, William [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai (China); Department of Surgery, University of British Columbia, Vancouver (Canada)], E-mail: wjia@interchange.ubc.ca

2008-10-24

21

Chikungunya virus capsid protein contains nuclear import and export signals  

PubMed Central

Background Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. Methods EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. Results Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin ? (Kar?) for its nuclear translocation. We also found that the Kar?4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. Conclusions We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export. PMID:23984714

2013-01-01

22

A cytoplasmically anchored nuclear protein interferes specifically with the import of nuclear proteins but not U1 snRNA  

PubMed Central

A cytoplasmically anchored mutant SV40 T antigen, FS T antigen, was shown previously to interfere specifically with the nuclear import of a heterologous nuclear protein, adenovirus 5 fiber protein, in cultured monkey cells (Schneider, J., C. Schindewolf, K. van Zee, and E. Fanning. 1988. Cell. 54:117-125; van Zee, K., F. Appel, and E. Fanning. 1991. Mol. Cell. Biol. 11:5137-5146). In this report, we demonstrate that FS T antigen also interferes with the nuclear import of adenovirus E1A and a peptide-albumin conjugate bearing multiple copies of the T antigen nuclear localization signal, but not with the import of U1 snRNA. A kinetic analysis indicates that nuclear import of the albumin- peptide conjugate is inhibited only when high intracellular concentrations of FS T antigen are reached. After microinjection into the cytoplasm of cultured cells, purified FS T antigen protein does not accumulate at the nuclear periphery, but rather is distributed in a punctate pattern throughout the cytoplasm. These data support a model in which cytoplasmic anchoring of FS T antigen enables the mutant protein to sequester and titrate out a cellular factor which is required for nuclear protein but not U1 snRNA import. PMID:8468344

1993-01-01

23

Identification and Characterization of Proteins Involved in Nuclear Organization Using Drosophila GFP Protein Trap Lines  

PubMed Central

Background Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern. Methodology/Principal Findings We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology. Conclusions/Significance These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins. PMID:23341925

Rohrbaugh, Margaret; Clore, Alyssia; Davis, Julia; Johnson, Sharonta; Jones, Brian; Jones, Keith; Kim, Joanne; Kithuka, Bramwel; Lunsford, Krystal; Mitchell, Joy; Mott, Brian; Ramos, Edward; Tchedou, Maza R.; Acosta, Gilbert; Araujo, Mark; Cushing, Stuart; Duffy, Gabriel; Graves, Felicia; Griffin, Kyler; Gurudatta, B. V.; Jackson, Deaundra; Jaimes, Denis; Jamison, Kendall; Jones, Khali; Kelley, Dhaujee; Kilgore, Marquita; Laramore, Derica; Le, Thuy; Mazhar, Bakhtawar; Mazhar, Muhammad M.; McCrary, Britney; Miller, Teanndras; Moreland, Celethia; Mullins, Alex; Munye, Elyas; Okoorie, Sheila; Pittman, Elisha; Roberts, Nikkita; Rose, De'Warren; Rowland, Alex; Shagarabi, Anwar; Smith, Jamela; Stallworth, Tayler; Stroud, Nicole; Sung, Elizabeth; Sung, Kai; Takenaka, Naomi; Torre, Eduardo; Veira, Jarvis; Vu, Kim; Wagstaff, William; Wood, Ashley M.; Wu, Karen; Yang, Jingping; Corces, Victor G.

2013-01-01

24

HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype  

PubMed Central

I. Summary Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed. PMID:19748605

Reeves, Raymond

2009-01-01

25

Nuclear localization signal binding proteins in higher plant nuclei.  

PubMed Central

The import of proteins into the nucleus is a vital process that is mediated by proteins which specifically recognize nuclear localization signals (NLSs). These factors have not been identified in plants. Previously, we demonstrated that higher plants possess a low-affinity binding site at the nuclear pore that specifically binds to several classes of functional NLSs. By the use of crosslinking reagents and a radiolabeled peptide to the bipartite NLS from the endogenous plant transcription factor Opaque2, two NLS binding proteins (NBPs) of 50-60 kDa and at least two NBPs of 30-40 kDa were identified. Competition studies indicated that labeling was specific for the functional NLS but not a mutant NLS impaired in vivo or a peptide unrelated to NLSs. Also, the apparent dissociation constant (100-300 microM) for labeling was similar to that of the binding site. Proteins of similar mass were labeled with two different crosslinking reagents, and concentration and time studies indicated that these NBPs were distinct proteins and not aggregates. Treatment with salt, detergent, or urea before or during NLS binding demonstrated that the properties of the binding site and the NBPs were identical. This tight correlation strongly indicates that some or all of the NBPs constitute the nuclear pore binding site. Overall, our results indicate that some components of NLS recognition are located at the nuclear pores in higher plants. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7846044

Hicks, G R; Raikhel, N V

1995-01-01

26

Generation of GTP-Ran for Nuclear Protein Import  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. Certain cytoplasmic proteins are actively transported into the nucleus through nuclear pores. In a paper in this week's issue the function of a participating soluble protein, p10, is elucidated (Nehrbass and Blobel, p. 120). Moore explains, in her Perspective, how these findings solve the riddle of the source of one of the molecules required for transport, GTP-Ran.

Mary Shannon Moore (Baylor College of Medicine;Department of Cell Biology)

1996-04-05

27

Prediction of nuclear proteins using nuclear translocation signals proposed by probabilistic latent semantic indexing  

PubMed Central

Background Identification of subcellular localization in proteins is crucial to elucidate cellular processes and molecular functions in a cell. However, given a tremendous amount of sequence data generated in the post-genomic era, determining protein localization based on biological experiments can be expensive and time-consuming. Therefore, developing prediction systems to analyze uncharacterised proteins efficiently has played an important role in high-throughput protein analyses. In a eukaryotic cell, many essential biological processes take place in the nucleus. Nuclear proteins shuttle between nucleus and cytoplasm based on recognition of nuclear translocation signals, including nuclear localization signals (NLSs) and nuclear export signals (NESs). Currently, only a few approaches have been developed specifically to predict nuclear localization using sequence features, such as putative NLSs. However, it has been shown that prediction coverage based on the NLSs is very low. In addition, most existing approaches only attained prediction accuracy and Matthew's correlation coefficient (MCC) around 54%~70% and 0.250~0.380 on independent test set, respectively. Moreover, no predictor can generate sequence motifs to characterize features of potential NESs, in which biological properties are not well understood from existing experimental studies. Results In this study, first we propose PSLNuc (Protein Subcellular Localization prediction for Nucleus) for predicting nuclear localization in proteins. First, for feature representation, a protein is represented by gapped-dipeptides and the feature values are weighted by homology information from a smoothed position-specific scoring matrix. After that, we incorporate probabilistic latent semantic indexing (PLSI) for feature reduction. Finally, the reduced features are used as input for a support vector machine (SVM) classifier. In addition to PSLNuc, we further identify gapped-dipeptide signatures for putative NLSs and NESs to develop a prediction method, PSLNTS (Protein Subcellular Localization prediction using Nuclear Translocation Signals). We apply PLSI to generate gapped-dipeptide signatures from both nuclear and non-nuclear proteins, and propose candidate sequence motifs for putative NLSs and NESs. Then, we incorporate only the proposed gapped-dipeptide signatures in an SVM classifier to mimic biological properties of NLSs and NESs for predicting nuclear localization in PSLNTS. Conclusions Experiment results demonstrate that the proposed method shows a significant improvement for nuclear localization prediction. To compare our predictive performance with other approaches, we incorporate two non-redundant benchmark data sets, a training set and an independent test set. Evaluated by five-fold cross-validation on the training set, PSLNuc attains an overall accuracy of 79.7%, which is 4.8% improvement over the state-of-the-art system. In addition, our method also enhances the MCC from 0.497 to 0.595. Compared on the independent test set, PSLNuc outperforms other predictors by 3.9%~19.9% on accuracy and 0.077~0.207 on MCC. This suggests that, in addition to NLSs, which have been shown important for nuclear proteins, NESs can also be an effective indicator to detect non-nuclear proteins. Most notably, using only a few proposed gapped-dipeptide signatures as input features for the SVM classifier, PSLNTS further enhances the accuracy and MCC to 80.9% and 0.618, respectively. Our results demonstrate that gapped-dipeptide signatures can better discriminate nuclear and non-nuclear proteins. Moreover, the proposed gapped-dipeptide signatures can be biologically interpreted and used in further experiment analyses of nuclear translocation signals, including NLSs and NESs. PMID:23282098

2012-01-01

28

Nuclear Accumulation of ?-Catenin Protein in Chemically Induced Rat Nephroblastomas  

PubMed Central

Wnt-signalling plays an important role in Wilms tumorigenesis. Upon activation, intracellular signal transduction results in stabilization, accumulation and nuclear translocation of ?-catenin. Nuclear ?-Catenin then acts in conjunction with members of the TCF/Lef family to cause transcriptional upregulation of specific proliferation-associated target genes such as c-myc or cyclin D1. Constitutive activation of ?-Catenin through mutations in CTNNB1 has been found in about 15% of Wilms tumors. Nuclear ?-catenin protein has been detected by immunohistochemistry in an even higher proportion of Wilms tumors suggesting alternative genetic pathways leading to ?-Catenin activation. Nephroblastomas induced in rats by either N-ethylnitrosourea or methyl(methoxymethyl)nitrosamine are histologically similar to Wilms tumors and provide a valuable rodent model. To study the involvement of the wnt-signalling pathway in rat nephroblastomas we examined 25 chemically induced rat nephroblastomas for nuclear accumulation of ?-Catenin protein and for mutations in Ctnnb1. 16 of 25 tumors showed nuclear accumulation of immunoreactive ?-catenin protein although no mutation was found in any of the tumors analyzed. These findings support the idea that active wnt-signalling contributes to tumorigenesis in carcinogen-induced nephroblastomas. PMID:19348510

Ehrlich, D; Bruder, E; Thome, MA; Gutt, CN; von Knebel Doeberitz, M; Niggli, F; Perantoni, AO; Koesters, R

2009-01-01

29

The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway  

SciTech Connect

The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

Kang, Won Kyung [Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198 (Japan)]. E-mail: wkkang@riken.jp; Kurihara, Masaaki [Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198 (Japan)]. E-mail: mkuri@riken.jp; Matsumoto, Shogo [Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198 (Japan)]. E-mail: smatsu@riken.jp

2006-06-20

30

New nuclear partners for nucleosome assembly protein 1: unexpected associations.  

PubMed

Histone chaperones are important players in chromatin dynamics. They are instrumental in nucleosome assembly and disassembly and in histone variant exchange reactions that occur during DNA transactions. The molecular mechanisms of their action are not well understood and may involve interactions with various protein partners in the context of the nucleus. In an attempt to further elucidate nuclear roles of histone chaperones, we performed a proteomic search for nuclear partners of a particular histone chaperone, nucleosome assembly protein 1 (Nap1). Proteins recognized as Nap1 partners by immuno-affinity capture and Far Western blots were identified by mass spectrometry. The identified partners are known to participate in a number of nuclear processes, including DNA replication, recombination, and repair as well as RNA transcription and splicing. Finding nuclear actin among the Nap1 partners may be of particular significance, in view of actin's role in transcription, transcription regulation, and RNA splicing. We are proposing a model of how actin-Nap1 interaction may be involved in transcription elongation through chromatin. In addition, awareness of the interactions between Nap1 and Hsp70, another identified partner, may help to understand nucleosome dynamics around sites of single-strand DNA break repair. These studies represent a starting point for further investigation of Nap1 associations in human cells. PMID:21102655

Seebart, Corrine; Prenni, Jessica; Tomschik, Miroslav; Zlatanova, Jordanka

2010-12-01

31

Identification and Characterization of the Ubiquitously Occurring Nuclear Matrix Protein NMP 238  

Microsoft Academic Search

By systematic comparison of two-dimensional electrophoretic patterns of nuclear matrix proteins an ubiquitously occurring (common) nuclear matrix protein, termed NMP 238, was detected. Localization of the protein in isolated nuclear matrices and in nuclear and cytoplasmic regions of cells was determined by confocal immunofluorescence microscopy. N-terminal protein sequencing, mass spectrometry, and sequencing of a human EST cDNA clone showed identity

Klaus Holzmann; Christopher Gerner; Thomas Korosec; Angelika Pöltl; Rudolf Grimm; Georg Sauermann

1998-01-01

32

Nuclear export of proteins and RNAs Sara Nakielny and Gideon Dreyfuss*  

E-print Network

420 Nuclear export of proteins and RNAs Sara Nakielny and Gideon Dreyfuss* Our understanding, signal-mediated export pathways. Nuclear export signals have been identified in several proteins, the majority of which are RNA-binding proteins. Nuclear export of RNA molecules is likely to be driven

Dreyfuss, Gideon

33

Nuclear envelope protein MAN1 regulates clock through BMAL1  

PubMed Central

Circadian clocks serve as internal pacemakers that influence many basic homeostatic processes; consequently, the expression and function of their components are tightly regulated by intricate networks of feedback loops that fine-tune circadian processes. Our knowledge of these components and pathways is far from exhaustive. In recent decades, the nuclear envelope has emerged as a global gene regulatory machine, although its role in circadian regulation has not been explored. We report that transcription of the core clock component BMAL1 is positively modulated by the inner nuclear membrane protein MAN1, which directly binds the BMAL1 promoter and enhances its transcription. Our results establish a novel connection between the nuclear periphery and circadian rhythmicity, therefore bridging two global regulatory systems that modulate all aspects of bodily functions. DOI: http://dx.doi.org/10.7554/eLife.02981.001 PMID:25182847

Lin, Shu-Ting; Zhang, Luoying; Lin, Xiaoyan; Zhang, Linda Chen; Garcia, Valentina Elizabeth; Tsai, Chen-Wei; Ptacek, Louis; Fu, Ying-Hui

2014-01-01

34

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes  

Microsoft Academic Search

An antibody was raised against “high mobility group” nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically

Reiner Westermann; Ulrich Grossbach

1984-01-01

35

Whole-genome screening identifies proteins localized to distinct nuclear bodies  

PubMed Central

The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies. PMID:24127217

Fong, Ka-wing; Li, Yujing; Wang, Wenqi; Ma, Wenbin; Li, Kunpeng; Qi, Robert Z.; Liu, Dan; Songyang, Zhou

2013-01-01

36

Nuclear protein IK undergoes dynamic subcellular translocation and forms unique nuclear bodies during the cell cycle  

PubMed Central

IK is a nuclear protein containing a unique domain named RED due to the presence of a repetitive arginine (R), aspartic (E), and glutamic acid (D) sequence. To date, the function of this protein remains largely unknown despite of a couple of previous studies in the literature. Here we report that depletion of IK via RNA interference results in mitotic arrest. We also demonstrate that IK undergoes dynamic translocation during interphase and mitosis. In particular, IK is primarily present in some interphase cells as nuclear foci/bodies which do not co-localize with nucleoli, PMA bodies and Cajal bodies. Pull-down analysis coupled with mass spectrometry reveals that IK is associated with DHX15, a putative ATP-dependent RNA helicase. Our results strongly suggest that IK may participate in pre-mRNA splicing and that it may be a useful biomarker for a new nuclear structure in the cell. PMID:24252166

2013-01-01

37

Protein Arginine Methylation in Candida albicans: Role in Nuclear Transport?  

PubMed Central

Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and ?-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3? strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2?/rmt2? strain, as well as biochemical evidence for additional cryptic PRMTs. PMID:17483287

McBride, Anne E.; Zurita-Lopez, Cecilia; Regis, Anthony; Blum, Emily; Conboy, Ana; Elf, Shannon; Clarke, Steven

2007-01-01

38

Properties of protein kinase C associated with nuclear membranes.  

PubMed Central

To study signal transduction directed towards the cell nucleus and at the nuclear membranes, we investigated the association of protein kinase C (PKC) with nuclear membranes obtained from nuclei isolated from bovine brain. By use of phorbol-ester-binding assays, significant amounts of PKC could be demonstrated in nuclei and nuclear membranes. Nuclear membranes are shown to be able to activate purified PKC. The PKC endogenously present in nuclear membranes appears to be a so-called 'membrane-inserted' form: it is permanently active, still binds phorbol ester, but its activity is no longer dependent on Ca2+ and cannot be activated by phorbol ester. On the other hand, this form of PKC can be inhibited by specific PKC inhibitors. By using histone HIIIS and a specific peptide substrate, it could be shown that after extraction with Triton X-100 the PKC can be stimulated by phospholipid again. Immunoblot analysis with isoenzyme-specific antibodies revealed that the alpha- and gamma-isoenzymes, but not the beta-isoenzyme, are associated with membranes derived from brain nuclei. Images Fig. 2. Fig. 6. PMID:1530569

Buchner, K; Otto, H; Hilbert, R; Lindschau, C; Haller, H; Hucho, F

1992-01-01

39

Further evidence that sperm nuclear proteins are necessary for embryogenesis.  

PubMed

We have recently presented evidence that the structural integrity of the mouse sperm nuclear matrix may be necessary for the proper unpackaging of sperm DNA for participation in embryogenesis. It is likely that the sperm nuclear matrix contributes to the organisation of the sperm DNA and its disturbance can seriously damage the paternal genome or its expression. In this work, we confirm our previous data and further suggest that even very subtle changes in the sperm nuclear structure may have a significant impact on embryo development. As reported previously, dithiothreitol (DTT) in the presence of an ionic detergent, ATAB, destabilized the nuclear matrix as measured by the halo assay, and oocytes injected with these nuclei failed to develop. We also discovered that omitting the protease inhibitor PMSF from the buffers used to extract spermatozoa prevented sperm injected into oocytes from participating in development. The organization of DNA into loop domains by the nuclear matrix in these nuclei appeared normal, as measured by the halo assay. Oocytes injected with sperm nuclei that had been washed with ATAB in the presence of phenylmethylsulphonyl fluoride (PMSF) but in the absence of DTT resulted in live births. Neither DTT treatment nor the absence of PMSF would be expected to disrupt the integrity of the paternal DNA. The data therefore suggest that even very subtle alterations in the structural proteins of the nucleus are enough to deprive sperm DNA of the ability to contribute to embryonic development. PMID:10840874

Ward, W S; Kishikawa, H; Akutsu, H; Yanagimachi, H; Yanagimachi, R

2000-02-01

40

Protein Sub-Nuclear Localization Prediction Using SVM and Pfam Domain Information  

PubMed Central

The nucleus is the largest and the highly organized organelle of eukaryotic cells. Within nucleus exist a number of pseudo-compartments, which are not separated by any membrane, yet each of them contains only a specific set of proteins. Understanding protein sub-nuclear localization can hence be an important step towards understanding biological functions of the nucleus. Here we have described a method, SubNucPred developed by us for predicting the sub-nuclear localization of proteins. This method predicts protein localization for 10 different sub-nuclear locations sequentially by combining presence or absence of unique Pfam domain and amino acid composition based SVM model. The prediction accuracy during leave-one-out cross-validation for centromeric proteins was 85.05%, for chromosomal proteins 76.85%, for nuclear speckle proteins 81.27%, for nucleolar proteins 81.79%, for nuclear envelope proteins 79.37%, for nuclear matrix proteins 77.78%, for nucleoplasm proteins 76.98%, for nuclear pore complex proteins 88.89%, for PML body proteins 75.40% and for telomeric proteins it was 83.33%. Comparison with other reported methods showed that SubNucPred performs better than existing methods. A web-server for predicting protein sub-nuclear localization named SubNucPred has been established at http://14.139.227.92/mkumar/subnucpred/. Standalone version of SubNucPred can also be downloaded from the web-server. PMID:24897370

Kumar, Ravindra; Jain, Sohni; Kumari, Bandana; Kumar, Manish

2014-01-01

41

Characterization of nuclear protein kinases of Xenopus laevis oocytes  

SciTech Connect

Xenopus laevis oocytes contain large nuclei (germinal vesicles) that can be isolated in very pure form and which permit the study of enzymatic activities present in these organelles. Incubation of pure oocyte nuclear homogenates with /sup 32/P in a buffered solution containing 5 mM MgCl/sub 2/ results in the phosphorylation of a large number of proteins by endogenous protein kinases. This phosphorylation is not affected by the addition of cyclic nucleotides or calcium ion and calmodulin. On the other hand the nuclear kinases are considerably stimulated by spermine and spermidine and strongly inhibited by heparin (10 ..mu..g/ml). Addition of exogenous protein substrates shows that the major oocyte kinases are very active with casein and phosvitin as substrates but do not phosphorylate histones or protamines. DEAE-Sephadex chromatography of the nuclear extract fractionates the casein phosphorylating activity in two main peaks. The first peak is not retained on the column equilibrated with 0.1 M NH/sub 2/SO/sub 4/ and uses exclusively ATP as phosphate donor and is insensitive to polyamines or heparin. The second peak which corresponds to 70% of the casein phosphorylation elutes at 0.27 M NH/sub 2/SO/sub 4/ and uses both ATP and GTP as phosphate donors and is greatly stimulated by polyamines and completely inhibited by 10 ..mu..g/ml heparin. On this evidence the authors conclude that the major protein kinase peak corresponds to casein kinase type II which has been found in mammalian nuclei.

Leiva, L.; Gonzalez, C.; Allende, C.; Allende, J.

1986-05-01

42

Analysis of hepatocyte nuclear factor-3 beta protein domains required for transcriptional activation and nuclear targeting.  

PubMed Central

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta and gamma) regulate transcription of the transthyretin (TTR) and numerous other liver-specific genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of developmental regulatory proteins including the Drosophila homeotic fork head (fkh) protein. The mammalian HNF-3/fkh family consists of at least thirty distinct members and is expressed in a variety of different cellular lineages. In addition to the winged helix motif, several HNF-3/fkh family members also share homology within transcriptional activation region II and III sequences. In the present study we further define the sequence boundaries of the HNF-3 beta N-terminal transcriptional activation domain to extend from amino acids 14 to 93 and include conserved region IV and V sequences. We also demonstrate that activity of the HNF-3 N-terminal domain was diminished by mutations which altered a putative alpha-helical structure located between amino acid residues 14 and 19. However, transcriptional activity was not affected by mutations which eliminated two conserved casein kinase I sites or increased the number of acidic amino acid residues in the N-terminal domain. Furthermore, we determined that the nuclear localization signal overlaps with the winged helix DNA-binding motif. These results suggest that conserved sequences within the winged helix motif of the HNF-3/fkh family may be involved not only in DNA recognition, but also in nuclear targeting. Images PMID:7739897

Qian, X; Costa, R H

1995-01-01

43

Nuclear Actin-Related Proteins in Epigenetic Control  

PubMed Central

The nuclear actin-related proteins (ARPs) share overall structure and low-level sequence homology with conventional actin. They are indispensable subunits of macromolecular machines that control chromatin remodeling and modification leading to dynamic changes in DNA structure, transcription, and DNA repair. Cellular, genetic, and biochemical studies suggest that the nuclear ARPs are essential to the epigenetic control of the cell cycle and cell proliferation in all eukaryotes, while in plants and animals they also exert epigenetic controls over most stages of multicellular development including organ initiation, the switch to reproductive development, and senescence and programmed cell death. A theme emerging from plants and animals is that in addition to their role in controlling the general compaction of DNA and gene silencing, isoforms of nuclear ARP-containing chromatin complexes have evolved to exert dynamic epigenetic control over gene expression and different phases of multicellular development. Herein, we explore this theme by examining nuclear ARP phylogeny, activities of ARP-containing chromatin remodeling complexes that lead to epigenetic control, expanding developmental roles assigned to several animal and plant ARP-containing complexes, the evidence that thousands of ARP complex isoforms may have evolved in concert with multicellular development, and ARPs in human disease. PMID:19766970

Meagher, Richard B.; Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen

2009-01-01

44

The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein  

Microsoft Academic Search

Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of

W. Matthew Michael; Paul S. Eder; Gideon Dreyfuss

1997-01-01

45

A Bayesian Network Model of Proteins' Association with Promyelocytic Leukemia (PML) Nuclear Bodies  

E-print Network

A Bayesian Network Model of Proteins' Association with Promyelocytic Leukemia (PML) Nuclear Bodies the complement of proteins associated with these intra-nuclear bodies, we construct a Bayesian network model%, the true positive rate is almost 50%, indicated by an independent nuclear proteome reference set. The model

Dellaire, Graham

46

Large-scale identification of mammalian proteins localized to nuclear sub-compartments  

Microsoft Academic Search

Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub- nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues

Heidi G. E. Sutherland; Gail K. Mumford; Kathryn Newton; Lisa V. Ford; Rachel Farrall; Graham Dellaire; Javier F. Cáceres; Wendy A. Bickmore

2001-01-01

47

NLS-tagging: an alternative strategy to tag nuclear proteins.  

PubMed

The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis. PMID:25260593

Giraud, Guillaume; Stadhouders, Ralph; Conidi, Andrea; Dekkers, Dick H W; Huylebroeck, Danny; Demmers, Jeroen A A; Soler, Eric; Grosveld, Frank G

2014-12-01

48

Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.  

PubMed

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus. PMID:2247074

Shaulsky, G; Goldfinger, N; Ben-Ze'ev, A; Rotter, V

1990-12-01

49

Reconstitution of nuclear protein transport with semi-intact yeast cells  

PubMed Central

We have developed an in vitro nuclear protein import reaction from semi- intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS- containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined. PMID:8227140

1993-01-01

50

Nuclear protein dysregulation in lymphoplasmacytic lymphoma/waldenstrom macroglobulinemia.  

PubMed

Waldenström macroglobulinemia (WM) is characterized by monoclonal gammopathy, usually IgM, in association with lymphoplasmacytic lymphoma (LPL). Little is known of the expression of nuclear proteins involved in B-cell development in LPL/WM. In this study, the expression patterns of PAX5/BSAP, MUM1/IRF4, and PRDM1/BLIMP1 were analyzed in plasma cells and lymphocytes in 29 cases of newly diagnosed LPL/WM by double immunohistochemical staining with CD138 and CD22. These patterns were compared with the expression profiles seen in normal bone marrow samples, reactive tonsils, and cases of plasma cell myeloma and marginal zone lymphoma. The median percentage of plasma cells coexpressing CD138 and PAX5 was significantly higher in LPL/WM compared with benign tissues (P = .001), marginal zone lymphoma (P = .002), and plasma cell myeloma (P < .0001), whereas the median percentage of plasma cells coexpressing CD138 and MUM1 was lower in LPL/WM than plasma cells in benign tissues (P = .02), marginal zone lymphoma (P = .001), and plasma cell myeloma (P = .0002). These findings show that a subset of plasma cells in LPL/WM demonstrates a nuclear protein expression pattern characteristic of the B-cell developmental program. Thus, the results better define the immunophenotypic profile of the neoplastic cells in LPL/WM. PMID:23355206

Roberts, Mark J; Chadburn, Amy; Ma, Shuo; Hyjek, Elizabeth; Peterson, LoAnn C

2013-02-01

51

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins  

PubMed Central

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV- 40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import. PMID:1332978

1992-01-01

52

Sorting of inner nuclear membrane-directed proteins at the endoplasmic reticulum membrane  

E-print Network

The current "diffusion-retention" model for protein trafficking to the inner nuclear membrane (INM) proposes that INM proteins diffuse laterally from the membrane of the endoplasmic reticulum into the INM and are then retained in the INM by binding...

Saksena, Suraj

2006-04-12

53

Diffusion Filters for Separation of Solvent-Protein and Protein-Protein Nuclear Overhauser Effects (HYDRA)  

E-print Network

(HYDRA) Gerhard Wider, Roland Riek, and Kurt Wu1thrich* Contribution from the Institut fu at different temperatures, is often employed. Here, we describe a novel 1D NMR difference experiment, HYDRA and the biological macromolecules.12 HYDRA contains strictly only water-protein interactions. For practical

Wider, Gerhard

54

Radioprotective Thiolamines WR-1065 and WR-33278 Selectively Denature Nonhistone Nuclear Proteins  

NASA Technical Reports Server (NTRS)

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca (2+) ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.

Booth, Valerie K.; Roberts, Jeanette C.; Warters, Raymond L.; Wilmore, Britta H.; Lepock, James R.

2000-01-01

55

Transcription-Dependent and Transcription-Independent Nuclear Transport of hnRNP Proteins  

E-print Network

. In mitosis, as the nuclear envelope breaks down, hnRNPs disperse throughout the cell. At the end of mitosis mitosis, after the nuclear enve- lope breaks down, they are found through- out the entire cellular spaceRNP proteins during mitosis, we compared the protein composition of hnRNPs purified from asynchronous

Dreyfuss, Gideon

56

Transcription signals of mitochondrial and nuclear genes for mitochondrial proteins in dicot plants  

Microsoft Academic Search

Mitochondria contain several large multisubunit enzyme complexes that are com- posed of proteins encoded in the nuclear and mitochondrial genomes. Particularly for correct assembly of these enzyme complexes, expression of the respective mitochondrial and nuclear genes has to be coordinated to ensure correct stoichi- ometries of the protein subunits. Part of this control and the response to specific demands is

A. Brennicke; E. Zabaleta; S. Dombrowski; M. Hoffmann; S. Binder

1999-01-01

57

Nuclear pore complex oxalate binding protein p62: expression in different kidney disorders  

Microsoft Academic Search

Background: Urolithiasis is a multifactorial process that starts with the formation of microcrystals in the urine and terminates as mature renal calculi. The oxalate binding protein plays a vital role in the transport of oxalate. The physiological significance of the presence of oxalate binding protein in the nuclear pore complex is not well understood. Methods: The nuclear envelope was extracted

P Sivakamasundari; P Kalaiselvi; R Sakthivel; R Selvam; P Varalakshmi

2004-01-01

58

Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins  

SciTech Connect

Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

2005-04-06

59

Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelod cell lines nuclear proteomes  

E-print Network

1 Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloïd cell lines nuclear proteomes Cécile Lelong(1)* , Mireille Chevallet(2) , Hélène Diemer(3) , Sylvie;2 Abstract One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance

Boyer, Edmond

60

The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein.  

PubMed Central

Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of this is the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein which, by virtue of its M9 domain, is actively exported from the nucleus and imported into the nucleus via a novel pathway mediated by the recently characterized transportin protein. Here we report that the shuttling hnRNP K protein contains a novel shuttling domain (termed KNS) which has many of the characteristics of M9, in that it confers bi-directional transport across the nuclear envelope. KNS-mediated nuclear import is dependent on RNA polymerase II transcription, and we show that a classical NLS can override this effect. Furthermore, KNS accesses a separate import pathway from either classical NLSs or M9. This demonstrates the existence of a third protein import pathway into the nucleus and thereby defines a new type of nuclear import/export signal. PMID:9218800

Michael, W M; Eder, P S; Dreyfuss, G

1997-01-01

61

Identification of nuclear proteins in soybean under flooding stress using proteomic technique.  

PubMed

Flooding stress restricts soybean growth, it results in decrease the production. In this report, to understand how nuclear proteins in soybean affected by flooding, abundance changes of those proteins was analyzed. Nuclear proteins were extracted from the root tips of soybean treated with or without flooding stress. The extracted proteins were analyzed using a label-free quantitative proteomic technique. Of a total of 94 nuclear proteins that were found to be responsive to flooding, the 19 and 75 proteins were increased and decreased, respectively. The identified flooding-responsive proteins were functionally classified, revealing that 8 increased proteins changed in protein synthesis, posttranslational modification, and protein degradation, while 34 decreased proteins were involved in transcription, RNA processing, DNA synthesis, and chromatin structure maintenance. Among these proteins, those whose levels changed more than 10 fold included two poly ADP-ribose polymerases and a novel G-domain-containing protein that might be involved in RNA binding. The mRNA expression levels of these three proteins indicated a similar tendency to their protein abundance changes. These results suggest that acceleration of protein poly-ADP-ribosylation and suppression of RNA metabolism may be involved in root tip of soybean under flooding stress. PMID:24237379

Oh, Myeong Won; Nanjo, Yohei; Komatsu, Setsuko

2014-05-01

62

ER Membrane Protein Complex Required for Nuclear Fusion Davis T.W. Ng and Peter Walter  

E-print Network

ER Membrane Protein Complex Required for Nuclear Fusion Davis T.W. Ng and Peter Walter Department is localized to the luminal (i.e., noncytoplasmic) face of the ER mem- brane, yet nuclear fusion must initiate of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear mem- brane fusion and requires ER

Walter, Peter

63

Sugar-dependent nuclear import of glycosylated proteins in living cells  

Microsoft Academic Search

The nuclear import of proteins larger than Mr 40,000 depends on the presence of a nuclear localization signal (NLS) corre- sponding either to a short peptide sequence or to defined sugars. The sugar-dependent nuclear import was previously evidenced by using glycosylated proteins (neoglycoproteins) introduced into the cytosol of cells either by electroporation or on digitonin-permeabilization and was shown to be

C. Rondanino; Annie-Claude Roche; Michel Monsigny

2003-01-01

64

Correlation of Nuclear Color and Opalescence with Protein S-thiolation in Human Lenses  

Microsoft Academic Search

Human lens nuclei were collected during routine cataract surgery and used to study the role of oxidation in cataract formation and brunescence. This study focused on the comparison of the intensities of nuclear opacity and pigmentation (brunescence) with the changes in free glutathione (GSH) and the three species of protein-thiol mixed disulfides: protein-S-S-glutathione (PSSG), protein-S S-cysteine (PSSC) and protein-S-S-?-glutamylcysteine (PSSGC).

MARJORIE F LOU; WILLIAM H TUNG; JOHN K WOLFE

1999-01-01

65

Biochemical investigations of the nuclear proteins during the transition of root meristems from quiescent to proliferating state in Pisum sativum  

Microsoft Academic Search

The electrophoretic analysis of nuclear proteins extracted from root meristems at different times of germination puts in evidence the variations of content of specific proteins. Several nuclear proteins are phosphorylated by endogenous protein kinase and often the maximum rate of phosphorylation it has been observed in proteins present in the nucleus at low concentrations. Moreover also the phosphorylation rate of

Donato Chiatante; Paola Brusa

1990-01-01

66

The nuclear import of ribosomal proteins is regulated by mTOR.  

PubMed

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A; Chen, Chien-Hung; Agarwal, Nitin K; Sarbassov, Dos D

2014-10-30

67

Adenovirus Ubiquitin-Protein Ligase Stimulates Viral Late mRNA Nuclear Export  

Microsoft Academic Search

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular

Jennifer L. Woo; Arnold J. Berk

2007-01-01

68

The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin  

Microsoft Academic Search

Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid

Deniz T Zeisig; Claudia B Bittner; Bernd B Zeisig; Maria-Paz García-Cuéllar; Jay L Hess; Robert K Slany

2005-01-01

69

A novel role for PA28-proteasome in nuclear speckle organization and SR protein trafficking  

E-print Network

1 A novel role for PA28-proteasome in nuclear speckle organization and SR protein trafficking degradation of key proteins. Analysis of 20S proteasome localization in human cell lines, using ectopic an alteration of intranuclear trafficking of SR proteins. Thus, our data identify proteasome-PA28 complexes

Paris-Sud XI, Université de

70

Quantitative determination of protein nuclear transport induced by phosphorylation or by proteolysis.  

PubMed

Nucleocytoplasmic transport of proteins in eukaryotic cells is a fundamental process for gene expression. The transport is regulated by posttranslational modifications of the proteins, such as ligand-binding, phosphorylation, and proteolysis. For monitoring the nuclear transport of proteins induced by a ligand binding, we have recently developed a genetically encoded bioluminescent indicator based on reconstitution of split fragments of Renilla reniformis (RLuc) by protein splicing with DnaE inteins. We herein describe that the technique is used for detecting phosphorylation- or proteolysis-induced nuclear transports of a target protein. Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation- and proteolysis-induced nuclear transport, respectively. Each STAT3 or SREBP-2 is connected with C-terminal halves of RLuc and DnaE. If the protein translocates into the nucleus, the C-terminal fragment of RLuc meets the N-terminal fragment of RLuc, and full-length RLuc is reconstituted by protein splicing in the nucleus. The indicator with SREBP-2 enabled us to quantify the intracellular concentrations of cholesterol. The indicator with STAT3 quantified the extent of the nuclear transport induced by representative cytokines. This simple assay based on protein nuclear transports allows the selection of suitable drugs among candidates and has significant potential for risk assessments, such as carcinogenic chemical screening in vitro and in vivo. PMID:16255591

Kim, Sung Bae; Takao, Ryohei; Ozawa, Takeaki; Umezawa, Yoshio

2005-11-01

71

Sequence and characterization of cytoplasmic nuclear protein import factor p97  

PubMed Central

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein. PMID:7615630

1995-01-01

72

Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated in transgenic tobacco  

E-print Network

Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated of the fusion protein was observed. By contrast, light-dependent nuclear import of the same fusion protein have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco

Schäfer, Eberhard

73

A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity  

PubMed Central

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro. PMID:2045410

1991-01-01

74

O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import  

PubMed Central

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component. PMID:1730755

1992-01-01

75

Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction*  

PubMed Central

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3–7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase II?. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions. PMID:21048195

Lewis, Aurélia E.; Sommer, Lilly; Arntzen, Magnus Ø.; Strahm, Yvan; Morrice, Nicholas A.; Divecha, Nullin; D'Santos, Clive S.

2011-01-01

76

Efficient plant male fertility depends on vegetative nuclear movement mediated by two families of plant outer nuclear membrane proteins.  

PubMed

Increasing evidence suggests that nuclear migration is important for eukaryotic development. Although nuclear migration is conserved in plants, its importance for plant development has not yet been established. The most extraordinary plant nuclear migration events involve plant fertilization, which is starkly different from that of animals. Instead of evolving self-propelled sperm cells (SCs), plants use pollen tubes to deliver SCs, in which the pollen vegetative nucleus (VN) and the SCs migrate as a unit toward the ovules, a fundamental but barely understood process. Here, we report that WPP domain-interacting proteins (WIPs) and their binding partners the WPP domain-interacting tail-anchored proteins (WITs) are essential for pollen nuclear migration. Loss-of-function mutations in WIT and/or WIP gene families resulted in impaired VN movement, inefficient SC delivery, and defects in pollen tube reception. WIPs are Klarsicht/ANC-1/Syne-1 Homology (KASH) analogs in plants. KASH proteins are key players in animal nuclear migration. Thus, this study not only reveals an important nuclear migration mechanism in plant fertilization but also, suggests that similar nuclear migration machinery is conserved between plants and animals. PMID:25074908

Zhou, Xiao; Meier, Iris

2014-08-12

77

System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics  

PubMed Central

The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum–inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane. PMID:21444689

Zuleger, Nikolaj; Kelly, David A.; Richardson, A. Christine; Kerr, Alastair R. W.; Goldberg, Martin W.; Goryachev, Andrew B.

2011-01-01

78

Identification of an unconventional nuclear localization signal in human ribosomal protein S2  

SciTech Connect

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-{beta}-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-{beta}-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric {beta}-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importin{beta} binding site fused to VP22 blocks nuclear import of rpS2-{beta}-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importin{alpha}/{beta} and transportin.

Antoine, M. [Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen, (Germany); Reimers, K. [Department for Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Podbielskistrasse 380, D-30659 Hannover, (Germany); Wirz, W. [Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen, (Germany); Gressner, A.M. [Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen, (Germany); Mueller, R. [Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen, (Germany); Kiefer, P. [Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen, (Germany)]. E-Mail: pkiefer@ukaachen.de

2005-09-16

79

Immunological identification and localization of the predominant nuclear protein of the amphibian oocyte nucleus  

PubMed Central

Nuclei of vitellogenic oocytes of the frog, Xenopus laevis, contain a prominent protein, representing about 10% of nuclear protein, which is characterized by a polypeptide of Mr 30,000. This protein is highly phosphorylated and acidic, displays several isoelectric variants with pI values ranging from 4.7 to 5.3, shows a high thermostability, is not stably complexed with other proteins, and is readily extracted in buffer solutions. Guinea pig antibodies against this protein have allowed its specific immunoprecipitation and localization by immunofluorescence microscopy, using both frozen tissue sections and cells grown in vitro. The protein is located almost exclusively in the nucleus where it appears to be spread throughout the nuclear interior. It is also a major nucleus-specific protein in vitellogenic and previtellogenic oocytes of other amphibian species as well as in other cell types, including hepatocytes, follicle epithelial cells, and cultured Xenopus cells, but is not detected in nuclei of transcriptionally inactive cells such as erythrocytes and spermatids. An immunologically related, if not identical, protein occurs in nuclei of higher vertebrate cells, including mammals. The properties of this abundant nuclear phosphoprotein and its possible relationship to the “nucleosome assembly factor” protein are discussed. It is suggested that this soluble protein serves a general nuclear function. Images PMID:6987661

Krohne, Georg; Franke, Werner W.

1980-01-01

80

Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane  

PubMed Central

Approximately 100 proteins are targeted to the inner nuclear membrane (INM), where they regulate chromatin and nuclear dynamics. The mechanisms underlying trafficking to the INM are poorly understood. The Caenorhabditis elegans SUN protein UNC-84 is an excellent model to investigate such mechanisms. UNC-84 recruits KASH proteins to the outer nuclear membrane to bridge the nuclear envelope (NE), mediating nuclear positioning. UNC-84 has four targeting sequences: two classical nuclear localization signals, an INM sorting motif, and a signal conserved in mammalian Sun1, the SUN—nuclear envelope localization signal. Mutations in some signals disrupt the timing of UNC-84 nuclear envelope localization, showing that diffusion is not sufficient to move all UNC-84 to the NE. Thus targeting UNC-84 requires an initial step that actively transports UNC-84 from the peripheral endoplasmic reticulum to the NE. Only when all four signals are simultaneously disrupted does UNC-84 completely fail to localize and to function in nuclear migration, meaning that at least three signals function, in part, redundantly to ensure proper targeting of UNC-84. Multiple mechanisms might also be used to target other proteins to the INM, thereby ensuring their proper and timely localization for essential cellular and developmental functions. PMID:21411627

Tapley, Erin C.; Ly, Nina; Starr, Daniel A.

2011-01-01

81

Characterization of a Drosophila phosphorylation-dependent nuclear-localization-signal-binding protein.  

PubMed Central

A 94 kDa nuclear-localization-signal (NLS)-binding protein was purified from Drosophila embryos. The NLS of the simian-virus-40 T-antigen is specifically bound by the dephosphorylated form of the protein. After phosphorylation, the affinity of the protein for the NLS is sharply decreased. In the dephosphorylated form, p94 (protein of 94 kDa) is the major NLS-binding protein in Drosophila embryos. Immunoprecipitation confirmed the ATP-dependent phosphorylation of p94, and co-precipitation of two additional phosphorylated proteins, indicated that the NLS-binding protein is part of a larger complex in Drosophila embryos. In agreement with the immunoprecipitation results, cross-linking experiments demonstrated the interaction of p94 with three additional proteins. These protein-protein interactions were also phosphorylation-dependent. PMID:9396726

Cserpan, I; Mathe, E; Patthy, A; Udvardy, A

1997-01-01

82

Nuclear Trafficking of Retroviral RNAs and Gag Proteins during Late Steps of Replication  

PubMed Central

Retroviruses exploit nuclear trafficking machinery at several distinct stages in their replication cycles. In this review, we will focus primarily on nucleocytoplasmic trafficking events that occur after the completion of reverse transcription and proviral integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which serves two essential roles: as the mRNA template for the translation of viral structural proteins and as the genome for encapsidation into virions. These full-length viral RNAs must overcome the cell’s quality control measures to leave the nucleus by co-opting host factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next, we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication. PMID:24253283

Stake, Matthew S.; Bann, Darrin V.; Kaddis, Rebecca J.; Parent, Leslie J.

2013-01-01

83

Heat-Shock Protein 90 Promotes Nuclear Transport of Herpes Simplex Virus 1 Capsid Protein by Interacting with Acetylated Tubulin  

PubMed Central

Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of ?-tubulin and Hsp90 interacted with the acetylated ?-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated ?-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance. PMID:24901434

Chen, Maoyun; Xiang, Yangfei; Jin, Fujun; Ma, Kaiqi; Qiu, Xianxiu; Wang, Qiaoli; Peng, Tao; Kitazato, Kaio; Wang, Yifei

2014-01-01

84

Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum  

PubMed Central

Background The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. Results We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. Conclusion Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology. PMID:23181666

2012-01-01

85

C. elegans Nuclear Envelope Proteins Emerin, MAN1, Lamin, and Nucleoporins Reveal Unique Timing of Nuclear Envelope Breakdown during Mitosis  

PubMed Central

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes. PMID:10982402

Lee, Kenneth K.; Gruenbaum, Yosef; Spann, Perah; Liu, Jun; Wilson, Katherine L.

2000-01-01

86

Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins.  

PubMed Central

Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vp1, carry individual nuclear targeting signals, Vp3(198-206) (Vp2(316-324) and Vp1(1-8), respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vp1 or Vp2/3, nuclear targeting-defective mutants thereof, Vp2/3(202T) and Vp1 delta N5, or nonoverlapping SV40 variants in which the genes for Vp1 and Vp2/3 are separated, and mutant derivatives of the gene carrying either one or both mutations. Nuclear targeting was assessed immunocytochemically following nuclear microinjection of the variant DNAs. When Vp2/3 and Vp1 mutants with defects in the nuclear targeting signals were expressed individually, the mutant proteins localized mostly to the cytoplasm. However, when mutant Vp2/3(202T) was coexpressed in the same cell along with wild-type Vp1, the mutant protein was effectively targeted to the nucleus. Likewise, the Vp1 delta N5 mutant protein was transported into the nucleus when wild-type Vp2/3 was expressed in the same cells. These results suggest that while Vp1 and Vp2/3 have independent nuclear targeting signals, additional signals, such as those defining protein-protein interactions, play a concerted role in nuclear localization along with the nuclear targeting signals of the individual proteins. Images PMID:7966613

Ishii, N; Nakanishi, A; Yamada, M; Macalalad, M H; Kasamatsu, H

1994-01-01

87

Proteomic Survey of Ubiquitin-Linked Nuclear Proteins in Interferon-Stimulated Macrophages  

PubMed Central

Ubiquitin modification plays a critical role in immune responses. Some cytoplasmic factors require ubiquitination to execute proper signaling upon pathogen and cytokine stimulation. However, ubiquitin modification and its functional significance have not been fully studied for many nuclear proteins. We report here that stimulation of RAW macrophages with interferon-? and toll-like receptor ligands that activates innate immune responses triggers a global increase in ubiquitinated proteins in the nucleus, pointing to the role for ubiquitin modification in regulating nuclear events during innate immune responses. By immunopurification and mass-spectrometry analyses, we found that more than 200 proteins are directly or indirectly associated with ubiquitin in stimulated RAW cells. These proteins included proteins in the ubiquitin pathways, those involved in DNA metabolism, chromatin and transcriptional regulation, and mRNA processing. The largest group of proteins found in our list was ribosomal proteins important for protein translation. Other proteins found here were heat shock proteins and stress-response factors, suggesting a link between macrophage activation and stress response. In conclusion, upon macrophage activation, a large number of nuclear proteins become associated with ubiquitin modification, presumably leading to a global shift in the genome activity, important for proper execution of innate immune responses. PMID:21428739

Kim, Ji Young; Anderson, Eric D.; Huynh, Walter; Dey, Anup

2011-01-01

88

Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C.  

PubMed

Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth. PMID:3163693

Fields, A P; Pettit, G R; May, W S

1988-06-15

89

Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy  

PubMed Central

Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein–coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy. PMID:23577669

Opella, Stanley J.

2014-01-01

90

KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion  

Microsoft Academic Search

KAR5 is required for membrane fusion dur- ing karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that

Christopher T. Beh; Valeria Brizzio; Mark D. Rose

1997-01-01

91

think proteins! think G-Biosciences! Enrichment of Nuclear, Mitochondrial, Cytosolic and  

E-print Network

transferred to a PVDF membrane, which was probed with antibodies specific for: caveolin, a membrane associatedthink proteins! think G-Biosciences! Enrichment of Nuclear, Mitochondrial, Cytosolic and Membrane. FOCUSTM SubCell kit enables the fast and easy isolation of nuclear, enriched mitochondrial, membrane

Lebendiker, Mario

92

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element  

SciTech Connect

Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s{sup -1}. We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

Mao, Grace [Department of Biomedical Engineering, University of California-Irvine, Irvine, CA 92697-2715 (United States); Brody, James P. [Department of Biomedical Engineering, University of California-Irvine, Irvine, CA 92697-2715 (United States)], E-mail: jpbrody@uci.edu

2007-11-09

93

Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?  

PubMed Central

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane- bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein- framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes. PMID:102650

1978-01-01

94

Deciphering apicoplast targeting signals – feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins  

Microsoft Academic Search

The malaria causing protozoan Plasmodium falciparum contains a vestigal, non-photosynthetic plastid, the apicoplast. Numerous proteins encoded by nuclear genes are targeted to the apicoplast courtesy of N-terminal extensions. With the impending sequence completion of an entire genome of the malaria parasite, it is important to have software tools in place for prediction of subcellular locations for all proteins. Apicoplast targeting

Jochen Zuegge; Stuart Ralph; Michael Schmuker; Geoffrey I. McFadden; Gisbert Schneider

2001-01-01

95

Rapid regulation of nuclear proteins by rapamycin-induced translocation in fission yeast.  

PubMed

Genetic analysis of protein function requires a rapid means of inactivating the gene under study. Typically, this exploits temperature-sensitive mutations or promoter shut-off techniques. We report the adaptation to Schizosaccharomyces pombe of the anchor-away technique, originally designed in budding yeast by Laemmli lab. This method relies on a rapamycin-mediated interaction between the FRB- and FKBP12-binding domains to relocalize nuclear proteins of interest to the cytoplasm. We demonstrate a rapid nuclear depletion of abundant proteins as proof of principle. PMID:24733494

Ding, Lin; Laor, Dana; Weisman, Ronit; Forsburg, Susan L

2014-07-01

96

Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress.  

PubMed

We have used a subcellular spatial razor approach based on LC-MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

Baqader, Noor O; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

2014-10-01

97

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein  

PubMed Central

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

98

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.  

PubMed

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

99

WNT Protein-independent Constitutive Nuclear Localization of ?-Catenin Protein and Its Low Degradation Rate in Thalamic Neurons*  

PubMed Central

Nuclear localization of ?-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that ?-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Cav3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying ?-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear ?-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with ?-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3?, components of the ?-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic ?-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of ?-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in ?-catenin labeling for ubiquitination and subsequent degradation. PMID:21757747

Misztal, Katarzyna; Wisniewska, Marta B.; Ambrozkiewicz, Mateusz; Nagalski, Andrzej; Kuznicki, Jacek

2011-01-01

100

Characterization of the nuclear localization signal of the mouse TET3 protein  

SciTech Connect

Highlights: •Amino acid sequence KKRK is responsible for nuclear localization of TET3. •Amino acid sequence KKRK are capable of targeting the cytoplasmic proteins to the nucleus. •Amino acid sequence KKRK are conserved in TET3 orthologs. -- Abstract: DNA demethylation is associated with gene activation and is mediated by a family of ten-eleven translocation (TET) dioxygenase. The TET3 protein is a 1668-amino-acid DNA demethylase that is predicted to possess five nuclear localization signals (NLSs). In this paper, we used a series of green fluorescent protein-tagged and mutation constructs to identify a conserved NLS (KKRK) embedded between amino acid 1615 and 1618 of mouse TET3. The KKRK sequence facilitates the cytoplasmic protein’s translocation into the nucleus. Additionally TET3 may be imported into the nucleus by importin-? and importin-?.

Xiao, Peng; Zhou, Xiao-long; Zhang, Hong-xiao; Xiong, Kai; Teng, Yun; Huang, Xian-ju; Cao, Rui; Wang, Yi; Liu, Hong-lin, E-mail: liuhonglin@263.net

2013-09-27

101

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus  

SciTech Connect

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China) [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Sun, Ya-Ni [College of Veterinary Medicine, Northwest A and F University, Shanxi, Yangling 712100 (China)] [College of Veterinary Medicine, Northwest A and F University, Shanxi, Yangling 712100 (China); Gao, Ji-Ming; Xie, Zhi-Jing [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China) [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Wang, Yu [Department of Basic Medical Sciences, Taishan Medical College, Shandong, Taian 271000 (China)] [Department of Basic Medical Sciences, Taishan Medical College, Shandong, Taian 271000 (China); Zhu, Yan-Li [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China) [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Jiang, Shi-Jin, E-mail: sjjiang@sdau.edu.cn [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China) [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China)

2013-02-05

102

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals  

NASA Technical Reports Server (NTRS)

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

103

Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain  

PubMed Central

Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins. PMID:17574289

Kakar, Mudit; Davis, James R.; Kern, Steve E.; Lim, Carol S.

2007-01-01

104

The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain.  

PubMed

The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization. PMID:15882967

Lechner, Mark S; Schultz, David C; Negorev, Dmitri; Maul, Gerd G; Rauscher, Frank J

2005-06-17

105

The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain  

SciTech Connect

The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

Lechner, Mark S. [Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 (United States)]. E-mail: msl27@drexel.edu; Schultz, David C. [Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 (United States); Negorev, Dmitri [Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 (United States); Maul, Gerd G. [Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 (United States); Rauscher, Frank J. [Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 (United States)

2005-06-17

106

Herpes Simplex Virus Infection Induces Phosphorylation and Delocalization of Emerin, a Key Inner Nuclear Membrane Protein?  

PubMed Central

The inner nuclear membrane (INM) contains specialized membrane proteins that selectively interact with nuclear components including the lamina, chromatin, and DNA. Alterations in the organization of and interactions with INM and lamina components are likely to play important roles in herpesvirus replication and, in particular, exit from the nucleus. Emerin, a member of the LEM domain class of INM proteins, binds a number of nuclear components including lamins, the DNA-bridging protein BAF, and F-actin and is thought to be involved in maintaining nuclear integrity. Here we report that emerin is quantitatively modified during herpes simplex virus (HSV) infection. Modification begins early in infection, involves multiple steps, and is reversed by phosphatase treatment. Emerin phosphorylation during infection involves one or more cellular kinases but can also be influenced by the US3 viral kinase, a protein whose function is known to be involved in HSV nuclear egress. The results from biochemical extraction analyses and from immunofluorescence of the detergent-resistant population demonstrate that emerin association with the INM significantly reduced during infection. We propose that the induction of emerin phosphorylation in infected cells may be involved in nuclear egress and uncoupling interactions with targets such as the lamina, chromatin, or cytoskeletal components. PMID:17301149

Morris, James B.; Hofemeister, Helmut; O'Hare, Peter

2007-01-01

107

Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.  

PubMed

E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT. PMID:24019486

Tsao, Hsiao-Wei; Tai, Tzong-Shyuan; Tseng, William; Chang, Hui-Hsin; Grenningloh, Roland; Miaw, Shi-Chuen; Ho, I-Cheng

2013-09-24

108

Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal  

PubMed Central

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response. PMID:24278431

Boisvert, Rebecca A.; Rego, Meghan A.; Azzinaro, Paul A.; Mauro, Maurizio; Howlett, Niall G.

2013-01-01

109

Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates.  

PubMed

Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies. PMID:16289535

Hübner, S; Eam, J E; Hübner, A; Jans, D A

2006-01-15

110

Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates  

SciTech Connect

Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.

Huebner, S. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, PO Box 13D, Monash University, Clayton, Victoria 3800 (Australia)]. E-mail: stefan.huebner@med.monash.edu.au; Eam, J.E. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, PO Box 13D, Monash University, Clayton, Victoria 3800 (Australia); Huebner, A. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, PO Box 13D, Monash University, Clayton, Victoria 3800 (Australia); Jans, D.A. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, PO Box 13D, Monash University, Clayton, Victoria 3800 (Australia)

2006-01-15

111

Multidimensional profiling of cell surface proteins and nuclear markers  

SciTech Connect

Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

2009-01-30

112

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor  

Microsoft Academic Search

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template.

Jamie T. Bridgham; Geeta N. Eick; Claire Larroux; Kirti Deshpande; Michael J. Harms; Marie E. A. Gauthier; Eric A. Ortlund; Bernard M. Degnan; Joseph W. Thornton

2010-01-01

113

Nuclear trafficking of proteins from RNA viruses: potential target for antivirals?  

PubMed

A key aspect of the infectious cycle of many viruses is the transport of specific viral proteins into the host cell nucleus to perturb the antiviral response. Examples include a number of RNA viruses that are significant human pathogens, such as human immunodeficiency virus (HIV)-1, influenza A, dengue, respiratory syncytial virus and rabies, as well agents that predominantly infect livestock, such as Rift valley fever virus and Venezuelan equine encephalitis virus. Inhibiting the nuclear trafficking of viral proteins as a therapeutic strategy offers an attractive possibility, with important recent progress having been made with respect to HIV-1 and dengue. The results validate nuclear protein import as an antiviral target, and suggest the identification and development of nuclear transport inhibitors as a viable therapeutic approach for a range of human and zoonotic pathogenic viruses. PMID:22750233

Caly, Leon; Wagstaff, Kylie M; Jans, David A

2012-09-01

114

Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo  

PubMed Central

Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt. PMID:16051845

Kurg, Reet; Sild, Kristiina; Ilves, Aigi; Sepp, Mari; Ustav, Mart

2005-01-01

115

Identification and Characterisation of a Nuclear Localisation Signal in the SMN associated protein, Gemin4  

PubMed Central

Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear /cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2–8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex. PMID:18675250

Lorson, Monique A.; Dickson, Alexa M.; Shaw, Debra J.; Todd, Adrian G.; Young, Elizabeth C.; Morse, Robert; Wolstencroft, Catherine; Lorson, Christian L.; Young, Philip J.

2013-01-01

116

Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4  

SciTech Connect

Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex.

Lorson, Monique A.; Dickson, Alexa M. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Shaw, Debra J.; Todd, Adrian G. [Institute of Biomedical and Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, St. Luke's Campus, Exeter, EX1 2LU (United Kingdom); Young, Elizabeth C. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Morse, Robert; Wolstencroft, Catherine [Institute of Biomedical and Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, St. Luke's Campus, Exeter, EX1 2LU (United Kingdom); Lorson, Christian L. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Young, Philip J. [Institute of Biomedical and Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, St. Luke's Campus, Exeter, EX1 2LU (United Kingdom)], E-mail: philip.young@pms.ac.uk

2008-10-10

117

R7BP: A Surprising New Link Between G Proteins, RGS Proteins, and Nuclear Signaling in the Brain  

NSDL National Science Digital Library

The regulators of G protein signaling (RGS proteins) bind directly to G protein alpha (Gα) subunits in brain and other tissues to determine the strength, duration, and fidelity of neurotransmitter receptor signaling. A recent study shows, quite unexpectedly, that one class of RGS proteins [the R7 subfamily bound to Gβ5 (R7-Gβ5)] shuttles between the plasma membrane and the nucleus with assistance from a novel shuttle protein, R7BP. R7BP binds directly to R7-Gβ5 and the protein complex is tethered to the plasma membrane by addition of a lipid, palmitate, on R7BP. Removal of palmitate results in the translocation of the R7BP–R7-Gβ5 complex to the nucleus, presumably for nontraditional signaling functions. These findings suggest an entirely novel mechanism for regulating neurotransmitter signaling. That is, R7BP transduces signals directly from receptors and G proteins at the plasma membrane to the nucleus, and this plasma membrane–nuclear shuttling is controlled by reversible palmitoylation of R7BP.

John R. Hepler (Emory University School of Medicine;Department of Pharmacology REV)

2005-07-26

118

Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein  

PubMed Central

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion. PMID:1370490

1992-01-01

119

Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells  

PubMed Central

Despite past progress in understanding mechanisms of cellular mechanotransduction, it is unclear whether a local surface force can directly alter nuclear functions without intermediate biochemical cascades. Here we show that a local dynamic force via integrins resulted in direct displacements of coilin and SMN proteins in Cajal bodies (CBs) and direct dissociation of coilin-SMN complexes. Spontaneous movements of coilin increased more than those of SMN in the same CB after dynamic force application. FRET changes of coilin-SMN depended on force magnitude, an intact F-actin, cytoskeletal tension, Lamin A/C, or substrate rigidity. Other protein pairs in CBs exhibited different magnitudes of FRET. Dynamic cyclic force induced tiny phase lags between various protein pairs in CBs, suggesting viscoelastic interactions between them. These findings demonstrate that dynamic force-induced direct structural changes of protein complexes in Cajal bodies may represent a unique mechanism of mechanotransduction that impacts on nuclear functions involved in gene expression. PMID:22643893

Poh, Yeh-Chuin; Shevtsov, Sergey P.; Chowdhury, Farhan; Wu, Douglas C.; Na, Sungsoo; Dundr, Miroslav; Wang, Ning

2012-01-01

120

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53  

SciTech Connect

Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

Sheren, Jamie E. [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States)] [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Department of Biology, Colorado State University, Fort Collins, CO 80523-1878 (United States)

2013-11-01

121

Effect of nitric oxide on gene transcription - S-nitrosylation of nuclear proteins  

PubMed Central

Nitric oxide (NO) plays an important role in many different physiological processes in plants. It mainly acts by post-translationally modifying proteins. Modification of cysteine residues termed as S-nitrosylation is believed to be the most important mechanism for transduction of bioactivity of NO. The first proteins found to be nitrosylated were mainly of cytoplasmic origin or isolated from mitochondria and peroxisomes. Interestingly, it was shown that redox-sensitive transcription factors are also nitrosylated and that NO influences the redox-dependent nuclear transport of some proteins. This implies that NO plays a role in regulating transcription and/or general nuclear metabolism which is a fascinating new aspect of NO signaling in plants. In this review, we will discuss the impact of S-nitrosylation on nuclear plant proteins with a focus on transcriptional regulation, describe the function of this modification and draw also comparisons to the animal system in which S-nitrosylation of nuclear proteins is a well characterized concept. PMID:23914201

Mengel, Alexander; Chaki, Mounira; Shekariesfahlan, Azam; Lindermayr, Christian

2013-01-01

122

Analysis of Nucleolar Protein Dynamics Reveals the Nuclear Degradation of Ribosomal Proteins  

PubMed Central

Summary Background The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased global analysis of the rates at which newly synthesized, endogenous proteins appear within mammalian nucleoli. Results Newly synthesized ribosomal proteins accumulated in nucleoli more quickly than other nucleolar components. Studies involving time-lapse fluorescence microscopy of stable HeLa cell lines expressing fluorescent-protein-tagged nucleolar factors also showed that ribosomal proteins accumulate more quickly than other components. Photobleaching and mass-spectrometry experiments suggest that only a subset of newly synthesized ribosomal proteins are assembled into ribosomes and exported to the cytoplasm. Inhibition of the proteasome caused an accumulation of ribosomal proteins in the nucleus but not in the cytoplasm. Inhibition of rRNA transcription prior to proteasomal inhibition further increased the accumulation of ribosomal proteins in the nucleoplasm. Conclusions Ribosomal proteins are expressed at high levels beyond that required for the typical rate of ribosome-subunit production and accumulate in the nucleolus more quickly than all other nucleolar components. This is balanced by continual degradation of unassembled ribosomal proteins in the nucleoplasm, thereby providing a mechanism for mammalian cells to ensure that ribosomal protein levels are never rate limiting for the efficient assembly of ribosome subunits. The dual time-lapse strategy used in this study, combining proteomics and imaging, provides a powerful approach for the quantitative analysis of the flux of newly synthesized proteins through a cell organelle. PMID:17446074

Lam, Yun Wah; Lamond, Angus I.; Mann, Matthias; Andersen, Jens S.

2007-01-01

123

Nuclear MYC Protein Overexpression is an Early Alteration in Human Prostate Carcinogenesis  

PubMed Central

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. By contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (e.g. Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically-defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression. PMID:18567993

Gurel, Bora; Iwata, Tsuyoshi; Koh, Cheryl; Jenkins, Robert B.; Lan, Fusheng; Van Dang, Chi; Hicks, Jessica L.; Morgan, James; Cornish, Toby C.; Sutcliffe, Siobhan; Isaacs, William B.; Luo, Jun; De Marzo, Angelo M.

2011-01-01

124

Tissue-Specific and Species-Specific Monoclonal Antibodies to Avian Red Cell Nuclear Proteins  

Microsoft Academic Search

In order to identify potential red cell-specific regulatory proteins and to define additional red cell-specific markers, we have isolated a series of hybridomas that produce monoclonal antibodies that react with nuclear preparations from avian red blood cells. Several antibodies have been well characterized for their tissue- and species-specific reactions by using solid-phase and protein-transfer radioimmunoassays as well as immunofluorescence. These

Caroline M. Kane; Pei Feng Cheng; John B. E. Burch; Harold Weintraub

1982-01-01

125

Interaction of HTLV1 Tax protein with calreticulin: Implications for Tax nuclear export and secretion  

Microsoft Academic Search

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy\\/tropical spastic paraparesis (HAM\\/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent

Timothy Alefantis; Katherine E. Flaig; Brian Wigdahl; Pooja Jain

2007-01-01

126

V-myc- and c-myc-encoded proteins are associated with the nuclear matrix.  

PubMed Central

A series of extraction procedures were applied to avian nuclei which allowed us to define three types of association of v-myc- and c-myc-encoded proteins with nuclei: (i) a major fraction (60 to 90%) which is retained in DNA- and RNA-depleted nuclei after low- and high-salt extraction, (ii) a small fraction (1%) released during nuclease digestion of DNA in intact nuclei in the presence of low-salt buffer, and (iii) a fraction of myc protein (less than 10%) extractable with salt or detergents and found to have affinity for both single- and double-stranded DNA. Immunofluorescence analysis with anti-myc peptide sera on cells extracted sequentially with nucleases and salts confirmed the idea that myc proteins were associated with a complex residual nuclear structure (matrix-lamin fraction) which also contained avian nuclear lamin protein. Dispersal of myc proteins into the cytoplasm was found to occur during mitosis. Both c-myc and v-myc proteins were associated with the matrix-lamin, suggesting that the function of myc may relate to nuclear structural organization. Images PMID:3872410

Eisenman, R N; Tachibana, C Y; Abrams, H D; Hann, S R

1985-01-01

127

SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY  

PubMed Central

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin ?, importin ?, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin ?, importin ?, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions. PMID:17056062

PARADISE, ALLISON; LEVIN, MIKHAIL K.; KORZA, GEORGE; CARSON, JOHN H.

2006-01-01

128

Dimerization and nuclear entry of mPER proteins in mammalian cells.  

PubMed

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner. PMID:10837028

Yagita, K; Yamaguchi, S; Tamanini, F; van Der Horst, G T; Hoeijmakers, J H; Yasui, A; Loros, J J; Dunlap, J C; Okamura, H

2000-06-01

129

Molecular Characterization of Three PRORP Proteins in the Moss Physcomitrella patens: Nuclear PRORP Protein Is Not Essential for Moss Viability  

PubMed Central

RNase P is a ubiquitous endonuclease that removes the 5? leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5?-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5?-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNAArg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNAArg (CCG) and pre-tRNAArg (UCU) but they cleaved pre-tRNAArg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical. PMID:25272157

Tanaka, Korechika; Kometani, Kazuki; Satoh, Hiroyuki; Sugita, Mamoru

2014-01-01

130

Archaeal RNase P has multiple protein subunits homologous to eukaryotic nuclear RNase P proteins.  

PubMed Central

Although archaeal RNase P RNAs are similar in both sequence and structure to those of Bacteria rather than eukaryotes, and heterologous reconstitution between the Bacillus subtilis RNase P protein and some archaeal RNase P RNAs has been demonstrated, no archaeal protein sequences with similarity to any known bacterial RNase P protein subunit have been identified, and the density of Methanothermobacter thermoautotrophicus RNase P in Cs2SO4 (1.42 g/mL) is inconsistent with a single small bacterial-like protein subunit. Four hypothetical open reading frames (MTH11, MTH687, MTH688, and MTH1618) were identified in the genome of M. thermoautotrophicus that have sequence similarity to four of the nine Saccharomyces cerevisiae RNase P protein subunits: Pop4p, Pop5p, Rpp1p, and Rpr2p, respectively. Polyclonal antisera generated to recombinant Mth11p, Mth687p, Mth688p, and Mth1618p each recognized a protein of the predicted molecular weight in western blots of partially purified M. thermoautotrophicus RNase P, and immunoprecipitated RNase P activity from the same partially purified preparation. RNase P in Archaea is therefore composed of an RNA subunit similar to bacterial RNase P RNA and multiple protein subunits similar to those in the eukaryotic nucleus. PMID:12003490

Hall, Thomas A; Brown, James W

2002-01-01

131

Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas  

E-print Network

of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies dem- onstrate that this protein is nuclear-localized under both nutrient- replete and is present in the biosphere as the oxidized anion, phosphate (Pi). Pi is not easily accessible to most plants

132

Comparison of nuclear matrix proteins between gastric cancer and normal gastric tissue  

Microsoft Academic Search

AIM: To study the alteration of nuclear matrix proteins (NMPs) in gastric cancer. METHODS: The NMPs extracted from 22 cases of gastric cancer and normal gastric tissues were investigated by SDS-PAGE technique and the data were analyzed using Genetools analysis software. RESULTS: Compared with normal gastric tissue, the expression of 30 ku and 28 ku NMPs in gastric cancer decreased

Qin-Xian Zhang; Yi Ding; Xiao-Ping Le; Wei Zhang; Ling Sun; Hui-Rong Shi

133

Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA  

E-print Network

Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA Megan by viruses or by transfection. Upon herpesviral in- fection of cells, the viral genome is chromatinized introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form

Knipe, David M.

134

Oxidized LDL affects smooth muscle cell growth through MAPK-mediated actions on nuclear protein import  

Microsoft Academic Search

Oxidized low density lipoprotein (oxLDL) plays an important role in the development of atherosclerosis partly through an action on cell proliferation and cell apoptosis. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell proliferation and apoptosis. The aim of this study was to determine if exposure of vascular smooth muscle cells (VSMC) to oxLDL affects

Mirna N. Chahine; David P. Blackwood; Elena Dibrov; Melanie N. Richard; Grant N. Pierce

2009-01-01

135

Characterization of the nuclear localization signal of high risk HPV16 E2 protein  

SciTech Connect

The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an {alpha} helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

Klucevsek, Kristin [Biology Department, Boston College, Higgins Hall, room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Wertz, Mary [Biology Department, Boston College, Higgins Hall, room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Lucchi, John [Biology Department, Boston College, Higgins Hall, room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Leszczynski, Anna [Biology Department, Boston College, Higgins Hall, room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Moroianu, Junona [Biology Department, Boston College, Higgins Hall, room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States)]. E-mail: moroianu@bc.edu

2007-03-30

136

The tight junction protein Z O-2 has several functional nuclear export signals  

SciTech Connect

The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.

Gonzalez-Mariscal, Lorenza [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico)]. E-mail: lorenza@fisio.cinvestav.mx; Ponce, Arturo [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico); Alarcon, Lourdes [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico); Jaramillo, Blanca Estela [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico)

2006-10-15

137

Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.  

PubMed Central

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells. Images PMID:6504045

Pekkala, D; Heath, B; Silver, J C

1984-01-01

138

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex  

PubMed Central

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport. PMID:20858414

Mattheyses, Alexa L.; Kampmann, Martin; Atkinson, Claire E.; Simon, Sanford M.

2010-01-01

139

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation  

SciTech Connect

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

Ostlund, Cecilia [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States) [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Guan, Tinglu [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States)] [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Figlewicz, Denise A. [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States)] [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Hays, Arthur P. [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States)] [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Worman, Howard J. [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States) [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Gerace, Larry [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States)] [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Schirmer, Eric C., E-mail: e.schirmer@ed.ac.uk [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR (United Kingdom)

2009-11-13

140

The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins  

PubMed Central

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins. PMID:11266464

Yoshida, Kimihisa; Blobel, Günter

2001-01-01

141

part of the spindle matrix in mitosis. Indeed, the yeast nuclear protein FIN1p contains coiled-  

E-print Network

part of the spindle matrix in mitosis. Indeed, the yeast nuclear protein FIN1p contains coiled- coil domains and associates with spindles during mitosis (46). Furthermore, purified FIN1p self to regulate many nuclear functions as well as nuclear structural integrity. At the onset of mitosis, lamins

Bermingham, Eldredge

142

Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.  

PubMed Central

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import. PMID:8670127

Seydel, U; Jans, D A

1996-01-01

143

Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction  

SciTech Connect

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

Gao, Feng-Hou [NO.3 People's Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China) [NO.3 People's Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China)] [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)] [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China)] [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China) [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

2009-11-15

144

Nuclear actin and actin-binding proteins in the regulation of transcription and gene expression  

PubMed Central

Nuclear actin is involoved in transcription of all three RNA polymerases, chromatin remodeling, and formation of hnRNP complexes as well as recruitment of histone modifier to the active gene. In addition, actin-binding proteins (ABPs) control actin nucleation, bundling, filament capping, fragmentation, and monomer availability in the cytoplasm. In recent years, more and more attention is on the role of actin and ABPs in the modulation of the subcellular localization of transcriptional regulators. This review focuses on the recent developments about transcription and transcriptional regulation by nuclear actin, regulation of muscle-specific gene expression, nuclear receptor and transcription complexes by ABPs. Among them, STARS and ABLIM regulate actin dynamics and SRF-dependent muscle-specific gene expression. Functionally and structurally unrelated cytoplasmic ABPs interact cooperatively with nuclear receptor and regulate its transactivation. Furthermore, ABPs also participate in the formation of transcription complexes. PMID:19459931

Zheng, Bin; Han, Mei; Bernier, Michel; Wen, Jin-kun

2010-01-01

145

Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins  

SciTech Connect

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.

Kumeta, Masahiro, E-mail: kumeta@lif.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kyoto 606-8501 (Japan); Hirai, Yuya; Yoshimura, Shige H. [Graduate School of Biostudies, Kyoto University, Kyoto 606-8501 (Japan); Horigome, Tsuneyoshi [Graduate School of Science and Technology, Niigata University, Niigata 950-2181 (Japan); Takeyasu, Kunio [Graduate School of Biostudies, Kyoto University, Kyoto 606-8501 (Japan)

2013-12-10

146

EBNA-5, an Epstein-Barr Virus-Encoded Nuclear Antigen, Binds to the Retinoblastoma and p53 Proteins  

Microsoft Academic Search

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long

Laszlo Szekely; Galina Selivanova; Kristinn P. Magnusson; George Klein; Klas G. Wiman

1993-01-01

147

Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-?/?.  

PubMed

The mechanism for nuclear envelope (NE) assembly is not fully understood. Importin-? and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process. Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-? and -? during NE assembly in Xenopus egg extracts. We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts, importin-? binds the chromatin NLS proteins rapidly. Meanwhile, importin-? binds cytoplasmic NE precursor membrane vesicles and nucleoporins. Through interacting with importin-? on the chromatin NLS proteins, importin-? targets the membrane vesicles and nucleoporins to the chromatin surface. Once encountering Ran-GTP on the chromatin generated by RCC1, importin-? preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly. NE assembly is disrupted by blocking the interaction between importin-? and NLS proteins with excess soluble NLS proteins or by depletion of importin-? from the extract. Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts. PMID:22847741

Lu, Quanlong; Lu, Zhigang; Liu, Qinying; Guo, Li; Ren, He; Fu, Jingyan; Jiang, Qing; Clarke, Paul R; Zhang, Chuanmao

2012-11-01

148

Proteomic Analysis of the Differential Protein Expression Reveals Nuclear GAPDH in Activated T Lymphocytes  

PubMed Central

Despite the important role of T cell activation in the adaptive immunity, very little is known about the functions of proteins that are differentially expressed in the activated T cells. In this study, we have employed proteomic approach to study the differentially expressed proteins in activated T cells. A total of 25 proteins was characterized that displayed a decreased expression, while a total of 20 proteins was characterized that displayed an increased expression in the activated T cells. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified unexpectedly as one of the up-regulated proteins. Western blot analysis of proteins separated by 2-dimensional gel electrophoresis had identified several modified GAPDHs which were detectable only in the activated T cells, but not in resting T cells. These modified GAPDHs had higher molecular mass and more basic PI, and were present in the nucleus of activated T cells. Promoter occupancy studies by chromatin immunoprecipitation assay revealed that nuclear GAPDH could be detected in the promoter of genes that were up-regulated during T cell activation, but not in the promoter of genes that were not unaffected or down-regulated. Our results suggest that nuclear GAPDH may function as transcriptional regulator in activated T cells. PMID:19621076

Sheng, Wei-Yun; Wang, Tzu-Chien V.

2009-01-01

149

SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown  

PubMed Central

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression. PMID:24662567

Turgay, Yagmur; Champion, Lysie; Balazs, Csaba; Held, Michael; Toso, Alberto; Gerlich, Daniel W.; Meraldi, Patrick

2014-01-01

150

Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein  

SciTech Connect

NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P{sup 27}KKRKAP{sup 276}) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

Kutty, R. Krishnan [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail: kuttyk@nei.nih.gov; Chen, Shanyi [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Samuel, William [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Vijayasarathy, Camasamudram [National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892 (United States); Duncan, Todd [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Tsai, Jen-Yue [Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Fariss, Robert N. [Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Carper, Deborah [Section on Molecular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Jaworski, Cynthia [Section on Molecular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Wiggert, Barbara [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

2006-07-14

151

MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors.  

PubMed

We have developed nanoparticles based on Murine Leukemia Virus virus-like-particles (VLPs) that efficiently deliver therapeutic bioactive proteins in their native state into target cells. Nuclear transcription factors and toxic proteins were incorporated into the VLPs from stable producer cells without transducing viral-encoded genetic material. Delivery of nuclear transcription factors required incorporation of nuclear export signals (NESs) into the vector backbone for the efficient formation of VLPs. In the presence of an appropriate targeting Env glycoprotein, transcription factors delivered and activated nuclear transcription in the target cells. Additionally, we show delivery of the bacterial toxin, MazF, which is an ACA-specific mRNA interferase resulted in the induction of cell death. The stable producer cells are protected from the toxin through co-expression of the anti-toxin MazE and continuously released MazF incorporating VLPs. This highly adaptable platform can be harnessed to alter and regulate cellular processes by bioactive protein delivery. PMID:24997480

Wu, Dai-Tze; Roth, Monica J

2014-09-01

152

Turnover of Amyloid Precursor Protein Family Members Determines Their Nuclear Signaling Capability  

PubMed Central

The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by ?-, ?-, and ?-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65. PMID:23874953

Trutzel, Annette; Bundschuh, Diana; Nitsch, Roger M.; Konietzko, Uwe

2013-01-01

153

Turnover of amyloid precursor protein family members determines their nuclear signaling capability.  

PubMed

The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by ?-, ?-, and ?-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65. PMID:23874953

Gersbacher, Manuel T; Goodger, Zoë V; Trutzel, Annette; Bundschuh, Diana; Nitsch, Roger M; Konietzko, Uwe

2013-01-01

154

Development of an ELISA detecting Tumor Protein 53-Induced Nuclear Protein 1 in serum of prostate cancer patients?  

PubMed Central

Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent “genome-keeper” p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage. PMID:24600558

Saadi, Houda; Seillier, Marion; Sandi, Maria Jose; Peuget, Sylvain; Kellenberger, Christine; Gravis, Gwenaelle; Dusetti, Nelson J.; Iovanna, Juan L.; Rocchi, Palma; Amri, Mohamed; Carrier, Alice

2013-01-01

155

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity  

SciTech Connect

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

Iwanaga, Kentaro [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Sueoka, Naoko [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Sato, Akemi [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Hayashi, Shinichiro [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan); Sueoka, Eisaburo [Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501 (Japan)]. E-mail: sueokae@post.saga-med.ac.jp

2005-08-05

156

Herpes simplex virus 2 UL13 protein kinase disrupts nuclear lamins  

SciTech Connect

Herpesviruses must cross the inner nuclear membrane and underlying lamina to exit the nucleus. HSV-1 US3 and PKC can phosphorylate lamins and induce their dispersion but do not elicit all of the phosphorylated lamin species produced during infection. UL13 is a serine threonine protein kinase conserved among many herpesviruses. HSV-1 UL13 phosphorylates US3 and thereby controls UL31 and UL34 nuclear rim localization, indicating a role in nuclear egress. Here, we report that HSV-2 UL13 alone induced conformational changes in lamins A and C and redistributed lamin B1 from the nuclear rim to intranuclear granular structures. HSV-2 UL13 directly phosphorylated lamins A, C, and B1 in vitro, and the lamin A1 tail domain. HSV-2 infection recapitulated the lamin alterations seen upon expression of UL13 alone, and other alterations were also observed, indicating that additional viral and/or cellular proteins cooperate with UL13 to alter lamins during HSV-2 infection to allow nuclear egress.

Cano-Monreal, Gina L.; Wylie, Kristine M.; Cao, Feng; Tavis, John E. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 South Grand Blvd., St. Louis, MO 63104 (United States); Morrison, Lynda A., E-mail: morrisla@slu.ed [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 South Grand Blvd., St. Louis, MO 63104 (United States)

2009-09-15

157

Acetylation of the SUN protein Mps3 by Eco1 regulates its function in nuclear organization  

PubMed Central

The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of certain types of persistent DNA double-stranded breaks. How these diverse SUN protein functions are regulated is unknown. Here we show that the Mps3 N-terminus is a substrate for the acetyltransferase Eco1/Ctf7 in vitro and in vivo and map the sites of acetylation to three lysine residues adjacent to the Mps3 transmembrane domain. Mutation of these residues shows that acetylation is not essential for growth, SPB duplication, or distribution in the nuclear membrane. However, analysis of nonacetylatable mps3 mutants shows that this modification is required for accurate sister chromatid cohesion and for chromosome recruitment to the nuclear membrane. Acetylation of Mps3 by Eco1 is one of the few regulatory mechanisms known to control nuclear organization. PMID:22593213

Ghosh, Suman; Gardner, Jennifer M.; Smoyer, Christine J.; Friederichs, Jennifer M.; Unruh, Jay R.; Slaughter, Brian D.; Alexander, Richard; Chisholm, Robert D.; Lee, Kenneth K.; Workman, Jerry L.; Jaspersen, Sue L.

2012-01-01

158

Sugar-dependent nuclear import of glycosylated proteins in living cells.  

PubMed

The nuclear import of proteins larger than Mr 40,000 depends on the presence of a nuclear localization signal (NLS) corresponding either to a short peptide sequence or to defined sugars. The sugar-dependent nuclear import was previously evidenced by using glycosylated proteins (neoglycoproteins) introduced into the cytosol of cells either by electroporation or on digitonin-permeabilization and was shown to be distinct from the peptide NLS-mediated pathway. In this work, we used a microinjection approach to compare the two nuclear import pathways in intact living cells. The intracellular localization of fluorescent NLS-BSA or Glc-BSA injected into the cytosol was analyzed by confocal microscopy. Novel differences between the two mechanisms were evidenced. First, Glc-BSA migrated less efficiently into the nucleus than NLS-BSA because of a cytosolic retention. Second, the import of neoglycoproteins was not affected by microinjection of antinuclear import factor importin/karyopherin beta antibodies, whereas the NLS-dependent transport was completely abolished. Third, the nuclear import activity of Glc-BSA was found to be cell cycle-dependent in thymidine and hydroxyurea-treated HeLa cells, with greatest efficiency during G1/S transition and S phases, whereas NLS-BSA was imported with the same efficiency during any stage of the cell cycle but the G2 phase. Fourth, we show that after mitosis, nonglycosylated BSA was excluded from the nucleus contrary to Glc-BSA. In both cases, the nuclear import signals (NLS or alpha-glucoside) were grafted onto BSA; such tools led to a clear-cut conclusion, which will reach a full physiological significance when they are confirmed in the case of endogenous (glyco)proteins. PMID:12672698

Rondanino, Christine; Bousser, Marie-Therese; Monsigny, Michel; Roche, Annie-Claude

2003-07-01

159

Protein Inhibitor of Activated STAT3 (PIAS3) Protein Promotes SUMOylation and Nuclear Sequestration of the Intracellular Domain of ErbB4 Protein*  

PubMed Central

ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function. PMID:22584572

Sundvall, Maria; Korhonen, Anna; Vaparanta, Katri; Anckar, Julius; Halkilahti, Kalle; Salah, Zaidoun; Aqeilan, Rami I.; Palvimo, Jorma J.; Sistonen, Lea; Elenius, Klaus

2012-01-01

160

Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1  

SciTech Connect

Oxidative stress can induce a covalent disulfide bond between protein and peptide thiols that is reversible through enzymatic catalysis. This process provides a post-translational mechanism for control of protein function and may also protect thiol groups from irreversible oxidation. High mobility group protein B1 (Hmgb1), a DNA-binding structural chromosomal protein and transcriptional co-activator was identified as a substrate of glutaredoxin. Hmgb1 contains 3 cysteines, Cys23, 45, and 106. In mild oxidative conditions, Cys23 and Cys45 readily form an intramolecular disulfide bridge, whereas Cys106 remains in the reduced form. The disulfide bond between Cys23 and Cys45 is a target of glutathione-dependent reduction by glutaredoxin. Endogenous Hmgb1 as well as GFP-tagged wild-type Hmgb1 co-localize in the nucleus of CHO cells. While replacement of Hmgb1 Cys23 and/or 45 with serines did not affect the nuclear distribution of the mutant proteins, Cys106-to-Ser and triple cysteine mutations impaired nuclear localization of Hmgb1. Our cysteine targeted mutational analysis suggests that Cys23 and 45 induce conformational changes in response to oxidative stress, whereas Cys106 appears to be critical for the nucleocytoplasmic shuttling of Hmgb1.

Hoppe, George [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States)]. E-mail: hoppeg@ccf.org; Talcott, Katherine E. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States); Bhattacharya, Sanjoy K. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States); Crabb, John W. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States); Sears, Jonathan E. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States)

2006-11-01

161

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs  

SciTech Connect

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

Lira, C.B.B. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil); Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Siqueira Neto, J.L. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil); Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Giardini, M.A. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil); Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Winck, F.V. [Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Ramos, C.H.I. [Instituto de Quimica, UNICAMP, Campinas, SP (Brazil); Cano, M.I.N. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil)]. E-mail: micano@ibb.unesp.br

2007-07-06

162

A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly  

PubMed Central

Within a single generation time a growing yeast cell imports ?14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. DOI: http://dx.doi.org/10.7554/eLife.03473.001 PMID:25144938

Schutz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Pena, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

2014-01-01

163

Apoptotic activity of a nuclear form of mitogaligin, a cell death protein  

SciTech Connect

Galig, an internal gene to the galectin-3 gene, encodes two proteins and induces cell death in human cells. Mitogaligin, one of these proteins, contains a mitochondrial targeting sequence and promotes the release of cytochrome c into the cytosol. Here, we show that mitogaligin can also localize to nucleus. The nuclear form of mitogaligin induced cell death through a pathway exhibiting typical properties of apoptosis. These observations indicate for the first time that mitogaligin expresses cytotoxic properties not only when addressed to mitochondria but also when targeted to the nucleus.

Gonzalez, Patrick; Robinet, Pauline; Charpentier, Stephane; Mollet, Lucile; Normand, Thierry; Dubois, Martine [Centre de Biophysique Moleculaire, (affiliated with University of Orleans) CNRS UPR4301, Rue Charles Sadron, 45071 Orleans Cedex 2 (France); Legrand, Alain [Centre de Biophysique Moleculaire, (affiliated with University of Orleans) CNRS UPR4301, Rue Charles Sadron, 45071 Orleans Cedex 2 (France)], E-mail: legrand@cnrs-orleans.fr

2009-01-23

164

Nuclear microinjection to assess how heterologously expressed proteins impact Ca2+ signals in Xenopus oocytes.  

PubMed

The Xenopus oocyte is frequently used for heterologous expression and for studying the spatiotemporal patterning of Ca(2+) signals. Here, we outline a protocol for nuclear microinjection of the Xenopus oocyte for the purpose of studying how subsequently expressed proteins impact intracellular Ca(2+) signals evoked by inositol trisphosphate (InsP3). Injected oocytes can easily be identified by reporter technologies and the impact of heterologously expressed proteins on the generation and properties of InsP3-evoked Ca(2+) signals can be resolved using caged InsP3 and fluorescent Ca(2+) indicators. PMID:23457340

Lin-Moshier, Yaping; Marchant, Jonathan S

2013-03-01

165

The human nuclear poly(a)-binding protein promotes RNA hyperadenylation and decay.  

PubMed

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAP? and PAP?, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAP?, redundantly with PAP?, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAP?/?, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression. PMID:24146636

Bresson, Stefan M; Conrad, Nicholas K

2013-01-01

166

The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay  

PubMed Central

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAP? and PAP?, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAP?, redundantly with PAP?, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAP?/?, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression. PMID:24146636

Bresson, Stefan M.; Conrad, Nicholas K.

2013-01-01

167

Importin 7 and Nup358 Promote Nuclear Import of the Protein Component of Human Telomerase  

PubMed Central

In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3?end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT. PMID:24586428

Frohnert, Cornelia; Hutten, Saskia; Wälde, Sarah; Nath, Annegret; Kehlenbach, Ralph H.

2014-01-01

168

Accumulation of the Inner Nuclear Envelope Protein Sun1 is Pathogenic in Progeric and Dystrophic Laminopathies  

PubMed Central

SUMMARY Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss Muscular Dystrophy (AD-EDMD) and Hutchinson-Gilford Progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna?/?) and progeroid Lmna?9 mutant mice are models for AD-EDMD and HGPS respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna?/? and Lmna?9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna?/? or Lmna?9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 over-accumulation in LMNA mutant fibroblasts and in HGPS cells corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna?/?, Lmna?9, and HGPS disorders. PMID:22541428

Chen, Chia-Yen; Chi, Ya-Hui; Mutalif, Rafidah Abdul; Starost, Matthew F.; Myers, Timothy G.; Anderson, Stasia A.; Stewart, Colin L.; Jeang, Kuan-Teh

2012-01-01

169

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11  

Microsoft Academic Search

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected

K. B. McElwain; J. E. Boynton; N. W. Gillham

1993-01-01

170

Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif  

Microsoft Academic Search

Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2\\/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized

Wenge Li; Siwang Yu; Tong Liu; Jung-Hwan Kim; Volker Blank; Hong Li; A.-N. Tony Kong

2008-01-01

171

Epstein-Barr Virus Nuclear Protein 2 is a Key Determinant of Lymphocyte Transformation  

Microsoft Academic Search

Epstein-Barr virus (EBV) efficiently transforms B lymphocytes to perpetual proliferation. The EBV laboratory strain P3HR-1 is transformation-incompetent and lacks a DNA segment that includes the EBV nuclear antigen 2 (EBNA-2) gene and a portion of the EBNA leader protein (EBNA-LP) gene. These two genes are expressed in transformed B lymphocytes. Recombinant transformation-competent EBVs were produced by transfecting P3HR-1-infected cells with

Jeffrey I. Cohen; Fred Wang; Joan Mannick; Elliott Kieff

1989-01-01

172

A Novel Set of Nuclear Localization Signals Determine Distributions of the ?CP RNA-Binding Proteins  

PubMed Central

?CPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major ?CP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each ?CP isoform. Immunofluorescence studies revealed three distinct distributions: ?CP1 and ?CP2 are predominantly nuclear with specific enrichment of ?CP1 in nuclear speckles, ?CP3 and ?CP4 are restricted to the cytoplasm, and ?CP2-KL, an ?CP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for ?CP isoforms. ?CP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in ?CP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in ?CP3 or ?CP4. Consistent with mapping studies, deletion of NLS I from ?CP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of ?CP2. These data demonstrate an unexpected complexity in the compartmentalization of ?CP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of ?CP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins. PMID:14612387

Chkheidze, Alexander N.; Liebhaber, Stephen A.

2003-01-01

173

The two tempos of nuclear pore complex evolution: highly adapting proteins in an ancient frozen structure  

PubMed Central

Background The origin of the nuclear compartment has been extensively debated, leading to several alternative views on the evolution of the eukaryotic nucleus. Until recently, too little phylogenetic information was available to address this issue by using multiple characters for many lineages. Results We analyzed 65 proteins integral to or associated with the nuclear pore complex (NPC), including all the identified nucleoporins, the components of their anchoring system and some of their main partners. We used reconstruction of ancestral sequences of these proteins to expand the detection of homologs, and showed that the majority of them, present all over the nuclear pore structure, share homologs in all extant eukaryotic lineages. The anchoring system, by contrast, is analogous between the different eukaryotic lineages and is thus a relatively recent innovation. We also showed the existence of high heterogeneity of evolutionary rates between these proteins, as well as between and within lineages. We show that the ubiquitous genes of the nuclear pore structure are not strongly conserved at the sequence level, and that only their domains are relatively well preserved. Conclusion We propose that an NPC very similar to the extant one was already present in at least the last common ancestor of all extant eukaryotes and it would not have undergone major changes since its early origin. Importantly, we observe that sequences and structures obey two very different tempos of evolution. We suggest that, despite strong constraints that froze the structural evolution of the nuclear pore, the NPC is still highly adaptive, modern, and flexible at the sequence level. PMID:16207356

Bapteste, Eric; Charlebois, Robert L; MacLeod, Dave; Brochier, Céline

2005-01-01

174

Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal.  

PubMed

The geminivirus transcription activation protein (TrAP) localizes to the nucleus and contains a putative nuclear localization signal (NLS) ((28)PRRRR(32)) on the N-terminus. The role of individual residues of this putative NLS on nuclear localization and symptom induction was investigated using TrAP of East African cassava mosaic Cameroon virus (EACMCV). Subcellular localization was conducted using the green fluorescent protein (GFP). Results showed that the proline residue at position 28 (Pro-28) is essential for nuclear localization whereas individually, none of the four contiguous arginines is necessary for nuclear targeting. The role of each of the five NLS amino acid residues on TrAP-mediated disease phenotype and gene silencing suppression was investigated by expressing these mutants in Nicotiana benthamiana from the PVX vector and under the control of the Cauliflower mosaic virus 35S promoter. Results showed that all five residues of the NLS play a role on disease phenotype production in N. benthamiana plants. Furthermore, each of the NLS residues appeared to be required for suppression of VIGS but appeared not to be required for the ability of TrAP to transactivate transcription and interact with adenosine kinase (ADK). PMID:19665038

Chowda-Reddy, R V; Dong, Wubei; Felton, Christian; Ryman, Danielle; Ballard, Keith; Fondong, Vincent N

2009-11-01

175

The SMN protein is a key regulator of nuclear architecture in differentiating neuroblastoma cells.  

PubMed

The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady-state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SMA). SMN is required in the cytoplasm for the addition of core, Sm, proteins to new snRNPs and is believed to accompany snRNPs to the CB. In most cell lines, gems are indistinguishable from CBs, although the structures are often separate in vivo. The relationship between CBs and gems is not fully understood, but there is evidence that symmetrical dimethylation of arginine residues in the CB protein coilin brings them together in HeLa cells. During neuronal differentiation of the human neuroblastoma cell line SH-SY5Y, CBs and gems increase their colocalization, mimicking changes seen during foetal development. This does not result from alterations in the methylation of coilin, but from increased levels of SMN. Expression of exogenous SMN results in an increased efficiency of snRNP transport to nuclear speckles. This suggests different mechanisms are present in different cell types and in vivo that may be significant for the tissue-specific pathology of SMA. PMID:19735367

Clelland, Allyson K; Kinnear, Nicholas P; Oram, Lisa; Burza, Julie; Sleeman, Judith E

2009-11-01

176

p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV- 40 T antigen proteins  

PubMed Central

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import. PMID:1659575

1991-01-01

177

Identification and characterization of a novel nuclear protein complex involved in nuclear hormone receptor-mediated gene regulation.  

PubMed

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of approximately 1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-alpha was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell. PMID:19131338

Garapaty, Shivani; Xu, Chong-Feng; Trojer, Patrick; Mahajan, Muktar A; Neubert, Thomas A; Samuels, Herbert H

2009-03-20

178

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization  

SciTech Connect

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X. [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of)] [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Lee, Kyung-Hoon [Department of Anatomy, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of) [Department of Anatomy, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Um, Sung Hee [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of) [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Kim, Jihoe [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of)] [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Ahn, Jee-Yin, E-mail: jeeahn@skku.edu [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of) [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of)

2012-01-15

179

The Drosophila nuclear lamina protein Otefin is required for germline stem cell survival  

PubMed Central

Summary LEM domain (LEM-D) proteins are components of an extensive protein network that assembles beneath the inner nuclear envelope. Defects in LEM-D proteins cause tissue-restricted human diseases associated with altered stem cell homeostasis. Otefin (Ote) is a Drosophila LEM-D protein that is intrinsically required for female germline stem cells (GSCs) maintenance. Previous studies linked Ote loss with transcriptional activation of the key differentiation gene, bag-of-marbles (bam), leading to the model that Ote tethers the bam gene to the nuclear periphery for gene silencing. Using genetic and phenotypic analyses of multiple ote?/? backgrounds, we obtained evidence that is inconsistent with this model. We show that bam repression is maintained in ote?/? GSCs and that germ cell loss persists in ote?/?, bam?/? mutants, together demonstrating that GSC loss is independent of bam transcription. We show the primary defect in ote?/? GSCs is a block of differentiation, which ultimately leads to germ cell death. PMID:23806619

Barton, Lacy J.; Pinto, Belinda S.; Wallrath, Lori L.; Geyer, Pamela K.

2013-01-01

180

Imaging of the DNA damage-induced dynamics of nuclear proteins via nonlinear photoperturbation.  

PubMed

Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at ? = 1050 nm in a 3D-confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two-photon photoactivation of PA-GFP fusion proteins at ? = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time-dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. PMID:23420601

Tomas, Martin; Blumhardt, Philipp; Deutzmann, Anja; Schwarz, Tobias; Kromm, Dimitri; Leitenstorfer, Alfred; Ferrando-May, Elisa

2013-08-01

181

Systematic Deletion and Mitotic Localization of the Nuclear Pore Complex Proteins of Aspergillus nidulans  

PubMed Central

To define the extent of the modification of the nuclear pore complex (NPC) during Aspergillus nidulans closed mitosis, a systematic analysis of nuclear transport genes has been completed. Thirty genes have been deleted defining 12 nonessential and 18 essential genes. Several of the nonessential deletions caused conditional phenotypes and self-sterility, whereas deletion of some essential genes caused defects in nuclear structure. Live cell imaging of endogenously tagged NPC proteins (Nups) revealed that during mitosis 14 predicted peripheral Nups, including all FG repeat Nups, disperse throughout the cell. A core mitotic NPC structure consisting of membrane Nups, all components of the An-Nup84 subcomplex, An-Nup170, and surprisingly, An-Gle1 remained throughout mitosis. We propose this minimal mitotic NPC core provides a conduit across the nuclear envelope and acts as a scaffold to which dispersed Nups return during mitotic exit. Further, unlike other dispersed Nups, An-Nup2 locates exclusively to mitotic chromatin, suggesting it may have a novel mitotic role in addition to its nuclear transport functions. Importantly, its deletion causes lethality and defects in DNA segregation. This work defines the dramatic changes in NPC composition during A. nidulans mitosis and provides insight into how NPC disassembly may be integrated with mitosis. PMID:16987955

Osmani, Aysha H.; Davies, Jonathan; Liu, Hui-Lin; Nile, Aaron

2006-01-01

182

The nuclear microspherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation protein in polyribosomal mRNPs from neurons.  

PubMed

The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation. PMID:16571602

Davidovic, Laetitia; Bechara, Elias; Gravel, Maud; Jaglin, Xavier H; Tremblay, Sandra; Sik, Attila; Bardoni, Barbara; Khandjian, Edouard W

2006-05-01

183

Evidence for Covalent Modification of the Nuclear Dot-associated Proteins PML and Sp100 by PIC1\\/SUMO1  

Microsoft Academic Search

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transforma- tion and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their ex- pression is enhanced by interferons (IFN). The recent finding that PIC1\\/SUMO-1, a

Thomas Sternsdorf; Kirsten Jensen; Hans Will

1997-01-01

184

Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3  

SciTech Connect

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada) [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

2012-10-10

185

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells  

PubMed Central

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell. PMID:6169730

1981-01-01

186

Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins  

Microsoft Academic Search

Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display

Liu Wang; Aihua Zheng; Ling Yi; Chongren Xu; Mingxiao Ding; Hongkui Deng

2004-01-01

187

Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast*  

PubMed Central

Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level. PMID:22685296

Sanchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M. Antonia; Soto, Teresa; Perez, Pilar; Gacto, Mariano; Cansado, Jose

2012-01-01

188

Heterodimerization with Small Maf Proteins Enhances Nuclear Retention of Nrf2 via Masking the NESzip Motif  

PubMed Central

Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized with the leucine zipper (ZIP) domain). Heterodimerization with MafG via ZIP-ZIP binding enhanced Nrf2 nuclear retention, which could be abrogated by the deletion of the ZIP domain or site-directed mutations targeting at the ZIP domain. In addition, dimerization with MafG precluded Nrf2zip/CRM1 binding, suggesting that Nrf2/MafG heterodimerization may simultaneously mask the NESzip motif. MafG-mediated nuclear retention may enable Nrf2 proteins to evade cytosolic proteasomal degradation and consequently stabilize Nrf2 signaling. For the first time, we show that at the physiological condition, the NESzip motif can be switched-off by heterodimerization. PMID:18585411

Li, Wenge; Yu, Siwang; Liu, Tong; Kim, Jung-Hwan; Blank, Volker; Li, Hong; Kong, A.-N. Tony

2008-01-01

189

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases  

PubMed Central

Summary X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in emerin. We validated one study, confirming that emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate emerin and a related protein, LAP2?, directly. Src phosphorylated emerin specifically at Y59, Y74 and Y95; the corresponding triple Y-to-F (`FFF') mutation reduced tyrosine phosphorylation by ?70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for emerin as a downstream effector and `signal integrator' for tyrosine kinase signaling pathway(s) at the nuclear envelope. PMID:19789182

Tifft, Kathryn E.; Bradbury, Katherine A.; Wilson, Katherine L.

2009-01-01

190

Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.  

PubMed

Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage. PMID:21160163

Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

2011-01-01

191

Physical modeling of the conformation of the unfolded proteins of the Nuclear Pore Complex  

NASA Astrophysics Data System (ADS)

Nuclear Pore Complex (NPC) is a biological ``nano-machine'' that controls the macromolecular transport between the cell nucleus and the cytoplasm. NPC functions without direct input of metabolic energy and without transitions of the gate from a ``closed'' to an ``open'' state during transport. The key and unique aspect of transport is the interaction of the transported molecules with the unfolded, natively unstructured proteins that cover the lumen of the NPC. Recently, the NPC inspired creation of artificial bio-mimetic for nano-technology applications. Although several models have been proposed, it is still not clear how the passage of the transport factors is coupled to the conformational dynamics of the unfolded proteins within the NPC. Morphology changes in assemblies of the unfolded proteins induced by the transport factors have been investigated experimentally in vitro. I will present a coarse-grained theoretical and simulation framework that mimics the interactions of unfolded proteins with nano-sized transport factors. The simple physical model predicts morphology changes that explain the recent puzzling experimental results and suggests possible new modes of transport through the NPC. It also provides insights into the physics of the behavior of unfolded proteins.

Zilman, Anton; Opferman, Michael; Coalson, Rob; Jasnow, David

2013-03-01

192

Physical and functional interactions of human papillomavirus E2 protein with nuclear receptor coactivators  

SciTech Connect

In addition to the human papillomavirus (HPV)-induced immortalization of epithelial cells, which usually requires integration of the viral DNA into the host cell genome, steroid hormone-activated nuclear receptors (NRs) are thought to bind to specific DNA sequences within transcriptional regulatory regions on the long control region to either increase or suppress transcription of dependent genes. In this study, our data suggest that the NR coactivator function of HPV E2 proteins might be mediated through physical and functional interactions with not only NRs but also the NR coactivators GRIP1 (glucocorticoid receptor-interacting protein 1) and Zac1 (zinc-finger protein which regulates apoptosis and cell cycle arrest 1), reciprocally regulating their transactivation activities. GRIP1 and Zac1 both were able to act synergistically with HPV E2 proteins on the E2-, androgen receptor-, and estrogen receptor-dependent transcriptional activation systems. GRIP1 and Zac1 might selectively function with HPV E2 proteins on thyroid receptor- and p53-dependent transcriptional activation, respectively. Hence, the transcriptional function of E2 might be mediated through NRs and NR coactivators to regulate E2-, NR-, and p53-dependent transcriptional activations.

Wu, M.-H. [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei City 114, Taiwan (China); Huang, C.-J. [Molecular Genetics and Biochemistry Laboratory, Cathay Medical Research Institute, Cathay General Hospital, Taipei County 221, Taiwan (China); Department of Biochemistry, National Defense Medical Center, Taipei City 114, Taiwan (China); Liu, S.-T. [Department of Biochemistry, National Defense Medical Center, Taipei City 114, Taiwan (China); Liu, P.-Y. [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei City 114, Taiwan (China); Ho, C.-L. [Division of Hematology/Oncology, Tri-Service General Hospital, National Defense Medical Center, Taipei City 114, Taiwan (China); Huang, S.-M. [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei City 114, Taiwan (China) and Department of Biochemistry, National Defense Medical Center, Taipei City 114, Taiwan (China)]. E-mail: shihming@ndmctsgh.edu.tw

2007-05-11

193

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.  

PubMed Central

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro. PMID:7799952

Gauthier-Rouviere, C; Vandromme, M; Lautredou, N; Cai, Q Q; Girard, F; Fernandez, A; Lamb, N

1995-01-01

194

Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein  

SciTech Connect

The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation.

Shen Weiping; Westgard, Elizabeth; Huang Liqun; Ward, Michael D.; Osborn, Jodi L.; Chau, Nha H.; Collins, Lindsay; Marcum, Benjamin; Koach, Margaret A.; Bibbs, Jennifer; Semmes, O. John [Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507 (United States); Kerry, Julie A. [Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507 (United States)], E-mail: kerryja@evms.edu

2008-06-20

195

Nuclear pore complex protein sequences determine overall copolymer brush structure and function.  

PubMed

The transport of cargo across the nuclear membrane is highly selective and accomplished by a poorly understood mechanism involving hundreds of nucleoporins lining the inside of the nuclear pore complex (NPC). Currently, there is no clear picture of the overall structure formed by this collection of proteins within the pore, primarily due to their disordered nature. We perform coarse-grained simulations of both individual nucleoporins and grafted rings of nups mimicking the in vivo geometry of the NPC and supplement this with polymer brush modeling. Our results indicate that different regions or blocks of an individual NPC protein can have distinctly different forms of disorder and that this property appears to be a conserved functional feature. Furthermore, this block structure at the individual protein level is critical to the formation of a unique higher-order polymer brush architecture that can exist in distinct morphologies depending on the effective interaction energy between the phenylalanine glycine (FG) domains of different nups. Because the interactions between FG domains may be modulated by certain forms of transport factors, our results indicate that transitions between brush morphologies could play an important role in regulating transport across the NPC, suggesting novel forms of gated transport across membrane pores with wide biomimetic applicability. PMID:24806932

Ando, David; Zandi, Roya; Kim, Yong Woon; Colvin, Michael; Rexach, Michael; Gopinathan, Ajay

2014-05-01

196

Association of protein kinase CK2 with nuclear matrix: influence of method of preparation of nuclear matrix.  

PubMed

Nuclear matrix (NM) plays a role of fundamental structural and functional significance as the site of replication, transcription, and RNA processing and transport, acting as an anchor or attachment site for a variety of enzymes and other proteins involved in these activities. We have previously documented that protein kinase CK2 translocates from the cytosol to the nucleus, where it associates preferentially with chromatin and NM, in response to certain growth stimuli. Considering that characteristics of the isolated NM can depend on the procedural employed for its isolation, we compared three standard methods for NM preparation to confirm the association of intrinsic CK2 with this structure. Our data suggest that the method used for isolating the NM can qualitatively influence the measurable NM-associated CK2. However, all three methods employed yielded qualitatively similar results with respect to the stimulus-mediated modulation of NM-associated CK2, thus further supporting the notion that NM is an important site for physiologically relevant functions of CK2. In addition, core filaments and cytoskeleton that were isolated by two of the preparative methods had a small but significant level of associated CK2 activity. PMID:9057107

Tawfic, S; Davis, A T; Faust, R A; Gapany, M; Ahmed, K

1997-03-01

197

Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase.  

PubMed

Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (LM) show both modifications act together to partially stabilize a short internal ?-helix comprising LM residues 43-46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48-67 into a new interface. The full structure of LM as bound to Ran (unlabeled) and Ran (216 aa) as bound by LM (unlabeled) places LM into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by LM binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:LM partner as an exportin, Crm1, or CAS. A model of Ran:LM:Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran's selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways. PMID:25331866

Bacot-Davis, Valjean R; Ciomperlik, Jessica J; Basta, Holly A; Cornilescu, Claudia C; Palmenberg, Ann C

2014-11-01

198

Yeast Importin-? (Srp1) Performs Distinct Roles in the Import of Nuclear Proteins and in Targeting Proteasomes to the Nucleus.  

PubMed

Srp1 (importin-?) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-? mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins. PMID:25274630

Chen, Li; Madura, Kiran

2014-11-14

199

Import and export of nuclear proteins: Focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor  

Microsoft Academic Search

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor from the cytoplasm to the nucleus.

Thomas Sebastian; S. Sreeja; Raghava Varman Thampan

2004-01-01

200

Nuclear import strategies of high-risk HPV18 L2 minor capsid protein.  

PubMed

We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap alpha2 adapter, and Kap beta2 and Kap beta3 nuclear import receptors. Moreover, binding of RanGTP to either Kap beta2 or Kap beta3 inhibits their interaction with L2, suggesting that these Kap beta/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap alpha2beta1 heterodimer and mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap beta2 and Kap beta3. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap alpha2beta1 heterodimers, Kap beta2 and Kap beta3. PMID:16733063

Klucevsek, K; Daley, J; Darshan, M S; Bordeaux, J; Moroianu, J

2006-08-15

201

Nuclear actin-related proteins at the core of epigenetic control.  

PubMed

Nuclear Actin-Related Proteins (ARPs) and actin combine as heterodimers to bind a large helicase subunit and form a core complex essential to the assembly and function of most chromatin remodeling and modifying machines. They are the most common shared subunits of these large and diverse assemblies in eukaryotes. We recently argued that most nuclear ARPs evolved directly from actin prior to the divergence of the eukaryotic kingdoms and did not evolve from pre-existing ARPs.2 Arabidopsis plants defective in nuclear ARP4, ARP5, ARP6, or ARP7 have extreme developmental phenotypes. Our recent publication demonstrates that ARP5-defective plants are not only dwarfed and have aberrant cell sizes, but are also hypersensitive to mutagenic agents that cause double strand DNA breaks.5 In Smith et al.6 we show that ARP6-defective plants, in addition to their extreme developmental phenotypes like small organs and early flowering, present an apparent "Phosphate Starvation Response" with strong morphological and molecular phenotypes. Herein, we interpret our latest data in the light of a hypothesis stating that in addition to their roles in overcoming DNA compaction that affects basal gene expression and silencing, nuclear ARP-containing chromatin complexes exert primary epigenetic control over high-level regulatory factors. PMID:21228632

Meagher, Richard B; Kandasamy, Muthugapatti K; Smith, Aaron P; McKinney, Elizabeth C

2010-05-01

202

HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358  

PubMed Central

Background Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells. Infection of non-dividing cells requires transport through the nuclear pore but how this is mediated is unknown. Results Here we present the crystal structure of the N-terminal capsid domain of HIV-1 in complex with the cyclophilin domain of nuclear pore protein NUP358. The structure reveals that HIV-1 is positioned to allow single-bond resonance stabilisation of exposed capsid residue P90. NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid. In contrast, the distantly related feline lentivirus FIV can bind NUP358 but is neither isomerized by it nor requires it for infection. Conclusion Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating. PMID:23902822

2013-01-01

203

Nuclear Import of Bovine Papillomavirus Type 1 E1 Protein Is Mediated by Multiple Alpha Importins and Is Negatively Regulated by Phosphorylation near a Nuclear Localization Signal?  

PubMed Central

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that the E1 protein interacted with importins ?3, ?4, and ?5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin ?3, ?4, or ?5 and other necessary import factors. We also observed that in vitro binding of E1 protein to all three ? importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type E1 protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin ? and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of nuclear E1 protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation. PMID:17192311

Bian, Xue-Lin; Rosas-Acosta, German; Wu, Yu-Chieh; Wilson, Van G.

2007-01-01

204

Nuclear dynamics of Arabidopsis calcium-dependent protein kinases in effector-triggered immunity.  

PubMed

Plants have evolved sophisticated innate immune systems to protect themselves from potential microbial invasions. Recognition of pathogen-derived virulence effector proteins is mediated by plant resistance (R) proteins and elicits potent defense responses, collectively termed as effector-triggered immunity (ETI). It has long been known that ETI is often accompanied with the increase of cytosolic Ca(2+) levels. We recently identified six closely related calcium-dependent protein kinases (CPKs) in Arabidopsis that orchestrate bifurcate ETI signaling via distinct substrate specificity and subcellular dynamics. In particular, the activation of CPK4, 5, 6 and 11 phosphorylates a specific subgroup of WRKY transcription factors to regulate transcriptional reprogramming crucial for restriction of pathogen growth. Upon ETI activation, a significant portion of CPK5 re-localizes to nucleus where it interacts and phosphorylates WRKY8, 28 and 48. Mass spectrometry analysis identified several conserved residues, including T247/T248 in WRKY48 and T199 in WRKY28 as the phosphorylation sites by CPKs. Here we reported that mutation of T198/T199 into alanine (TT198AA) in WRKY28 completely abolished its phosphorylation by CPK4 and 11. The importance of nuclear localization of CPK5 was further demonstrated by that CPK5 fused with nuclear export signal abolished its synergistic effect with WRKY8, 28 and 48 on the activation of defense gene. In contrast, effector AvrRpt2 likely functions in the cytoplasm to activate the transcriptional reprogramming of defense genes, consistent with the plasma membrane localization of its RPS2 receptor. Our data established WRKYs as bona fide substrates of CPKs and provided a framework for the study of CPK-WRKY cascade in diverse biological processes. Our results also demonstrated that the nuclear localization and subcellular dynamics of CPKs are essential to relay distinct ETI signaling events. PMID:23425856

Gao, Xiquan; He, Ping

2013-04-01

205

Oxidative stress impairs nuclear proteins binding to the insulin responsive element in the GLUT4 promoter  

Microsoft Academic Search

.\\u000a Aims\\/hypothesis:   Substantial evidence suggests an important role for the expression of GLUT4 in adipocytes, in the pathogenesis of insulin\\u000a resistance and Type II (non-insulin-dependent) diabetes mellitus. We investigated whether oxidative stress decreases GLUT4 expression by impairing DNA binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter. \\u000a \\u000a \\u000a \\u000a Methods:   3T3-L1 adipocytes were exposed to micromolar H2O2 concentrations

D. Pessler; A. Rudich; N. Bashan

2001-01-01

206

Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila.  

PubMed

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids. PMID:14970188

Yu, Jiangtao; Fleming, Shawna L; Williams, Byron; Williams, Erika V; Li, ZeXiao; Somma, Patrizia; Rieder, Conly L; Goldberg, Michael L

2004-02-16

207

FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know?  

PubMed Central

The binding of nuclear proteins to chromatin in live cells has been analyzed by kinetic modeling procedures applied to experimental data from fluorescence recovery after photobleaching (FRAP). The kinetic models have yielded a number of important biological predictions about transcription, but concerns have arisen about the accuracy of these predictions. First, different studies using different kinetic models have arrived at very different predictions for the same or similar proteins. Second, some of these divergent predictions have been shown to arise from technical issues rather than biological differences. For confidence and accuracy, gold standards for the measurement of in vivo binding must be established by extensive cross validation using both different experimental methods and different kinetic modeling procedures. PMID:20413286

Mueller, Florian; Mazza, Davide; Stasevich, Timothy J.; McNally, James G.

2010-01-01

208

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication  

PubMed Central

The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication. PMID:22345442

Cao, Shuai; Liu, Xiaoling; Yu, Maorong; Li, Jing; Jia, Xiaojuan; Bi, Yuhai; Sun, Lei; Gao, George F.

2012-01-01

209

The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

Nakagawa, Koji, E-mail: k_nakagawa@pharm.hokudai.ac.jp [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan)] [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan)] [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan) [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan) [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan) [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)

2012-11-02

210

Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.  

PubMed

Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-?) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-? expression was evaluated. Both TNF-? mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-? promoter. In the presence of NEP the activity of TNF-? promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-? promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-? promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-? promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-? expression. PMID:24657783

Lara-Sampablo, Alejandra; Flores-Alonso, Juan Carlos; De Jesús-Ortega, Nereyda; Santos-López, Gerardo; Vallejo-Ruiz, Verónica; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Herrera-Camacho, Irma; Reyes-Leyva, Julio

2014-06-24

211

Expression of Nuclear Factor Erythroid 2 Protein in Malignant Cutaneous Tumors  

PubMed Central

Background Reactive oxygen species (ROS) damages cell molecules, and modifies cell signaling. The nuclear factor E2-related factor (Nrf2) is a critical transcription regulator, which protects cells against oxidative damage. Nrf2 expression is increased in a large number of cancers. However, little information has been reported regarding the expression of Nrf2 in skin cancers. Hence, we explored the expression of Nrf2 protein in skin cancers. Methods The Nrf2 protein expression in 24 specimens, including 6 malignant melanomas (MM), 6 squamous cell carcinomas (SCC), 6 basal cell carcinomas (BCC), and 6 normal skin tissues, was evaluated by western blotting. Immunohistochemical staining was performed. The expression of Kelch-like ECH-associated protein 1 (Keap1), the key regulator of Nrf2, was also analyzed by western blotting. Results Small interfering RNA transfection to the melanoma cell line G361 confirmed that an approximately 66 kDa band was the true Nrf2 band. The western blot revealed that the Nrf2 protein was definitely expressed in normal skin tissues, but the Nrf2 expression was decreased in MM, SCC, and BCC. Immunohistochemical examination showed that expression of Nrf2 was decreased in all skin cancer tissues compared to the normal skin tissues. Keap1 was not expressed in all malignant skin tumors and normal skin tissues by western blot. Conclusions ROS was increased in various types of cancers which proteins were highly expressed or underexpressed. This study demonstrated that the expression of Nrf2 protein was down-regulated in human malignant skin tumors. We suggest that decreased expression of Nrf2 is related to skin cancers.

Choi, Chang Yong; Kim, Jin Young; Wee, Seo Yeong; Lee, Jang Hyun; Nam, Doo Hyun; Cho, Moon Kyun; Lee, Yoon Jin; Nam, Hae Seon; Lee, Sang Han; Cho, Sung Woo

2014-01-01

212

Inducibility of the HS II enhancer depends on binding of an erythroid specific nuclear protein.  

PubMed Central

An erythroid specific, inducible enhancer associated with hypersensitive site II (HS II) plays a central role in the function of the human beta globin dominant control region. The HS II enhancer consists of tandem AP-1 binding sites and has been shown to bind members of the ubiquitous jun and fos families of proteins. The same sites are now shown to bind the erythroid specific protein, NF-E2. Inducibility of the HS II enhancer depends on NF-E2 binding, even in the presence of another hypersensitive site. Further, increased activity of the enhancer in induced K562 cells correlates with the presence of NF-E2, which appears to be present in a modified form. NF-E2 is distinct from some enhancer binding proteins in K562 nuclear extracts, in that it does not contain Fos or Fra-1 protein. Thus, binding by NF-E2 may be the mechanism, whereby tandem AP-1 binding sites confer erythroid specificity on the HS II enhancer. Images PMID:2235483

Ney, P A; Sorrentino, B P; Lowrey, C H; Nienhuis, A W

1990-01-01

213

Identification and characterization of two trypanosome TFIIS proteins exhibiting particular domain architectures and differential nuclear localizations  

PubMed Central

Nuclear transcription of Trypanosoma brucei displays unusual features. Most protein-coding genes are organized in large directional gene clusters, which are transcribed polycistronically by RNA polymerase II (pol II) with subsequent processing to generate mature mRNA. Here, we describe the identification and characterization of two trypanosome homologues of transcription elongation factor TFIIS (TbTFIIS1 and TbTFIIS2-1). TFIIS has been shown to aid transcription elongation by relieving arrested pol II. Our phylogenetic analysis demonstrated the existence of four independent TFIIS expansions across eukaryotes. While TbTFIIS1 contains only the canonical domains II and III, the N-terminus of TbTFIIS2-1 contains a PWWP domain and a domain I. TbTFIIS1 and TbTFIIS2-1 are expressed in procyclic and bloodstream form cells and localize to the nucleus in similar, but distinct, punctate patterns throughout the cell cycle. Neither TFIIS protein was enriched in the major pol II sites of spliced-leader RNA transcription. Single RNA interference (RNAi)-mediated knock-down and knockout showed that neither protein is essential. Double knock-down, however, impaired growth. Repetitive failure to generate a double knockout of TbTFIIS1 and TbTFIIS2-1 strongly suggests synthetical lethality and thus an essential function shared by the two proteins in trypanosome growth. PMID:18627464

Uzureau, Pierrick; Daniels, Jan-Peter; Walgraffe, David; Wickstead, Bill; Pays, Etienne; Gull, Keith; Vanhamme, Luc

2008-01-01

214

Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons  

SciTech Connect

Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.

Young, Kevin G. [Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, K1H 8L6 (Canada); University of Ottawa Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario (Canada); Kothary, Rashmi [Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, K1H 8L6 (Canada); University of Ottawa Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario (Canada); Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario (Canada); Department of Medicine, University of Ottawa, Ottawa, Ontario (Canada)], E-mail: rkothary@ohri.ca

2008-09-10

215

lin-8, Which Antagonizes Caenorhabditis elegans Ras-Mediated Vulval Induction, Encodes a Novel Nuclear Protein That Interacts With the LIN-35 Rb Protein  

PubMed Central

Ras-mediated vulval development in C. elegans is inhibited by the functionally redundant sets of class A, B, and C synthetic Multivulva (synMuv) genes. Three of the class B synMuv genes encode an Rb/DP/E2F complex that, by analogy with its mammalian and Drosophila counterparts, has been proposed to silence genes required for vulval specification through chromatin modification and remodeling. Two class A synMuv genes, lin-15A and lin-56, encode novel nuclear proteins that appear to function as a complex. We show that a third class A synMuv gene, lin-8, is the defining member of a novel C. elegans gene family. The LIN-8 protein is nuclear and can interact physically with the product of the class B synMuv gene lin-35, the C. elegans homolog of mammalian Rb. LIN-8 likely acts with the synMuv A proteins LIN-15A and LIN-56 in the nucleus, possibly in a protein complex with the synMuv B protein LIN-35 Rb. Other LIN-8 family members may function in similar complexes in different cells or at different stages. The nuclear localization of LIN-15A, LIN-56, and LIN-8, as well as our observation of a direct physical interaction between class A and class B synMuv proteins, supports the hypothesis that the class A synMuv genes control vulval induction through the transcriptional regulation of gene expression. PMID:16020796

Davison, Ewa M.; Harrison, Melissa M.; Walhout, Albertha J. M.; Vidal, Marc; Horvitz, H. Robert

2005-01-01

216

A nuclear hormone receptor-associated protein that inhibits transactivation by the thyroid hormone and retinoic acid receptors.  

PubMed Central

Nuclear hormone receptors are transcription factors that require multiple protein-protein interactions to regulate the expression of their target genes. Using the yeast two-hybrid system, we identified a protein, thyroid hormone receptor uncoupling protein (TRUP), that specifically interacts with a region of the human thyroid hormone receptor (TR) consisting of the hinge region and the N-terminal portion of the ligand binding domain in a hormone-independent manner. Interestingly, TRUP inhibits transactivation by TR and the retinoic acid receptor but has no effect on the estrogen receptor or the retinoid X receptor in mammalian cells. We also demonstrate that TRUP exerts its action on TR and retinoic acid receptor by interfering with their abilities to interact with their DNA. TRUP represents a type of regulatory protein that modulates the transcriptional activity of a subclass of the nuclear hormone receptor superfamily by preventing interaction with their genomic response elements. Images Fig. 1 Fig. 2 Fig. 3 PMID:7568167

Burris, T P; Nawaz, Z; Tsai, M J; O'Malley, B W

1995-01-01

217

The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane.  

PubMed

In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain-containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1-Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1-Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB. PMID:24515347

Chen, Jingjing; Smoyer, Christine J; Slaughter, Brian D; Unruh, Jay R; Jaspersen, Sue L

2014-02-17

218

Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae.  

PubMed

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells. PMID:10952997

Yun, C Y; Fu, X D

2000-08-21

219

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs  

PubMed Central

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth. PMID:1706724

1991-01-01

220

Brd4-Mediated Nuclear Retention of the Papillomavirus E2 Protein Contributes to Its Stabilization in Host Cells  

PubMed Central

Papillomavirus E2 is a multifunctional viral protein that regulates many aspects of the viral life cycle including viral episome maintenance, transcriptional activation, and repression. E2 is degraded by the ubiquitin-proteasome pathway. Cellular bromodomain protein Brd4 has been implicated in the stabilization of the E2 protein. E2 normally shuttles between the cytoplasm and the nucleus. In this study, we demonstrate that E2 ubiquitylation mostly occurs in the cytoplasm. We also find that the interaction with Brd4 promotes nuclear retention of papillomavirus E2 proteins and contributes to their stabilization in the nucleus. Compared to wild type E2 proteins, nuclear-localization-defective mutants are rapidly degraded by the ubiquitin-proteasome pathway; however, co-expression of Brd4 redirects these mutants into the nucleus and significantly increases their stability. We further demonstrate that tethering E2 proteins to chromatin as either double-bromodomain fusion proteins or histone 2B (H2B) fusion proteins significantly stabilizes the E2 proteins. Our studies suggest that chromatin recruitment of the E2 protein via interaction with Brd4 prevents E2 ubiquitylation and proteasomal degradation in the cytoplasm, leading to its stabilization in the nucleus. These studies bring new insights for understanding Brd4-mediated E2 stabilization, and provide an additional mechanism by which the chromatin-associated Brd4 regulates E2 functions. PMID:24448221

Li, Jing; Li, Qing; Diaz, Jason; You, Jianxin

2014-01-01

221

Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections.  

PubMed

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized. PMID:23117894

Eberhart, Anja; Kimura, Hiroshi; Leonhardt, Heinrich; Joffe, Boris; Solovei, Irina

2012-10-01

222

Nuclear Fragile X Mental Retardation Protein Is localized to Cajal Bodies  

PubMed Central

Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome. PMID:24204304

Tremblay, Sandra; Rose, Timothy M.; Cote, Jocelyn; De Koninck, Paul; Khandjian, Edouard W.

2013-01-01

223

Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus  

PubMed Central

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization. PMID:11533202

Goodin, Michael M.; Austin, Jennifer; Tobias, Renee; Fujita, Miki; Morales, Christina; Jackson, Andrew O.

2001-01-01

224

Viral Immediate-Early Proteins Abrogate the Modification by SUMO1 of PML and Sp100 Proteins, Correlating with Nuclear Body Disruption  

Microsoft Academic Search

PML nuclear bodies (NBs) are subnuclear structures whose integrity is compromised in certain human dis- eases, including leukemia and neurodegenerative disorders. Infection by a number of DNA viruses similarly triggers the reorganization of these structures, suggesting an important role for the NBs in the viral infection process. While expression of the adenovirus E4 ORF3 protein leads to only a moderate

STEFAN MULLER; ANNE DEJEAN

1999-01-01

225

The PSI family of nuclear proteins is required for growth in arabidopsis.  

PubMed

PSI1 was identified as a gene that is co-expressed with the phytosulfokine (PSK) receptor genes PSKR1 and PSKR2 in Arabidopsis thaliana. It represents a plant-specific protein family of unknown function with six members in two clades. Clade 1 members PSI1, PSI2 and PSI3 were characterized in this study. All three are nuclear localized. A predicted N-terminal myristoylation site was functionally analyzed. psi1-1 seedlings have shorter roots and hypocotyls. This growth-retarded phenotype was restored by expression of either wildtype PSI1 or PSI1 G2A with a mutated myristate attachment site in the psi1-1 background suggesting that myristate attachment was not essential for PSI1 function. psi2-1 and psi3-1 seedlings have a wildtype phenotype but overexpression of PSI1 or PSI2 promoted seedling growth. PSI2 activity appears to be linked to PSK signaling as psi2-1 and psi2-1 psi3-1 roots are unresponsive to PSK. PSI3 functions in vegetative plant growth synergistic with PSI2. psi3-1 and particularly psi2-1 psi3-1 rosettes are small. Overexpression of PSI3 promoted plant growth indicating that PSI3 is limiting at the vegetative stage. Severe dwarfism of psi2-1 psi3-1 plants results from reduced cell growth and proliferation and premature leaf growth arrest. Plants further display reduced fertility and premature senescence revealing a crucial function of PSI proteins in vegetative growth and reproduction. Psi single and double knock-out plants have less and PSI3ox plants have more starch compared to wt and growth retardation is partially rescued by sucrose. Our studies reveal a crucial function of the nuclear-localized PSI proteins in growth possibly through metabolic control. PMID:25062973

Stührwohldt, Nils; Hartmann, Jens; Dahlke, Renate I; Oecking, Claudia; Sauter, Margret

2014-10-01

226

A Nuclear Factor of High Mobility Group Box Protein in Toxoplasma gondii  

PubMed Central

High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-?, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host–parasite interactions for T. gondii infection. PMID:25369210

Wang, Hui; Lei, Tao; Liu, Jing; Li, Muzi; Nan, Huizhu; Liu, Qun

2014-01-01

227

Arrest of Nuclear Division in Plasmodium through Blockage of Erythrocyte Surface Exposed Ribosomal Protein P2  

PubMed Central

Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6–8 hrs during early schizogony, starting around 26–28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80–85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division. PMID:22912579

Das, Sudipta; Basu, Himanish; Korde, Reshma; Tewari, Rita; Sharma, Shobhona

2012-01-01

228

Antigenic differences in nuclear proteins of normal liver and hepatoma. Identification of a nuclear protein present in hepatocytes but absent in hepatoma cells  

Microsoft Academic Search

Two main types of proteins are associated with chromatin. The histones are a well characterized group of basic proteins, which seem to be involved in the general organization of the chromatin. The nonhistone proteins (NHP) ~ are a much more heterogeneous group of proteins and are believed to be involved in the regulation of gene expression (1, 2). If gene

ERKKI RUOSLAHTI; EVA ENGVALL; HANNU JALANKO; DAVID E. COMINGS

1977-01-01

229

Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins  

PubMed Central

The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between the PML SUMO-interacting motif (SIM) and SUMO, but instead results from oxidation-mediated PML multimerization. Oxidized PML spherical meshes recruit UBC9, which enhances PML sumoylation, allow partner recruitment through SIM interactions, and ultimately enhance partner sumoylation. Intermolecular SUMO–SIM interactions then enforce partner sequestration within the NB inner core. Accordingly, oxidative stress enhances NB formation and global sumoylation in vivo. Some NB-associated sumoylated partners also become polyubiquitinated by RNF4, precipitating their proteasomal degradation. As several partners are protein-modifying enzymes, NBs could act as sensors that facilitate and confer oxidative stress sensitivity not only to sumoylation but also to other post-translational modifications, thereby explaining alterations of stress response upon PML or NB loss. PMID:24637324

Sahin, Umut; Ferhi, Omar; Jeanne, Marion; Benhenda, Shirine; Berthier, Caroline; Jollivet, Florence; Niwa-Kawakita, Michiko; Faklaris, Orestis; Setterblad, Niclas; Lallemand-Breitenbach, Valerie

2014-01-01

230

A role for timely nuclear translocation of clock repressor proteins in setting circadian clock speed.  

PubMed

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

Lee, Euna; Kim, Eun Young

2014-09-01

231

Flowering and genome integrity control by a nuclear matrix protein in Arabidopsis.  

PubMed

The matrix attachment regions (MARs) binding proteins could finely orchestrate temporal and spatial gene expression during development. In Arabidopsis, transposable elements (TEs) and TE-like repeat sequences are transcriptionally repressed or attenuated by the coordination of many key players including DNA methyltransferases, histone deacetylases, histone methyltransferases and the siRNA pathway, which help to protect genomic integrity and control multiple developmental processes such as flowering. We have recently reported that an AT-hook nuclear matrix binding protein, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), participates in a histone deacetylation (HDAC) complex to silence TEs and genes containing a TE-like sequence, including AtMu1, FWA and FLOWERING LOCUS C (FLC) in Ler background. We have shown that TEK knockdown causes increased histone acetylation, reduced H3K9me2 and moderate reduction of DNA methylation in the target loci, leading to the de-repression of FLC and FWA, as well as TE reactivation. Here we discuss the role of TEK as a putative MAR binding protein which functions in the maintenance of genome integrity and in flowering control by silencing TEs and repeat-containing genes. PMID:23836195

Xu, Yifeng; Gan, Eng-Seng; He, Yuehui; Ito, Toshiro

2013-01-01

232

A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed  

PubMed Central

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins.

Lee, Euna

2014-01-01

233

Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p  

PubMed Central

The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors. PMID:24297441

Pierce, Jacqueline B.; van der Merwe, George

2014-01-01

234

A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein  

Microsoft Academic Search

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev\\/Rex

L. K. Venkatesh; T. Gettemeier; G. Chinnadurai

2003-01-01

235

Phorbol ester-mediated association of protein kinase C to the nuclear fraction in NIH 3T3 cells.  

PubMed

Treatment of intact NIH 3T3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid redistribution (stabilization) of protein kinase C to the particulate fraction. Part of the enzyme activity stabilized to the membrane fraction in response to TPA can be recovered associated with nuclear-cytoskeletal components. An apparently pure nuclear fraction prepared from NIH 3T3 cells was found to contain 25-30% of the total membrane-associated protein kinase C activity when isolated in the presence of Ca2+. In untreated control cells, most of this activity found with the nuclear fraction can be extracted by chelators. Phorbol ester (TPA) treatment of NIH 3T3 cells induces the tight association of protein kinase C to the nucleus; this tightly bound activity is not dissociable by chelators and can be recovered only by solubilization with detergent. Nuclei purified from untreated human promyelocytic leukemic HL-60 cells contain higher amounts of chelator-stable, detergent-extractable protein kinase C activity compared with control NIH 3T3 cells. However, TPA treatment of HL-60 cells does not enhance the amount of protein kinase C found tightly associated with the nuclear fraction. Immunohistochemical studies with polyclonal antibodies directed against protein kinase C further indicate that TPA treatment of NIH 3T3 cells does significantly enhance the amount of protein kinase C found tightly associated with the nucleus and cytoskeleton, whereas exposure of HL-60 cells to TPA does not appreciably alter the amount of protein kinase C observed to be associated with the nuclear fraction. The TPA-mediated association (activation) of protein kinase C to the nuclear and cytoskeletal fractions with NIH 3T3 cells is further supported by the enhanced phosphorylation of specific endogenous proteins noted when purified nuclei and cytoskeletal preparations are incubated with [gamma-32P]ATP. These results suggest that tumor promoters may induce association (activation) of protein kinase C with different subcellular components to alter the availability of endogenous substrates. This may result in differential responses by different cell types during exposure to tumor promoters. PMID:3127041

Thomas, T P; Talwar, H S; Anderson, W B

1988-04-01

236

Assemblons: nuclear structures defined by aggregation of immature capsids and some tegument proteins of herpes simplex virus 1.  

PubMed Central

In cells infected with herpes simplex virus 1 (HSV-1), the viral proteins ICP5 (infected-cell protein 5) and VP19c (the product of UL38) are associated with mature capsids, whereas the same proteins, along with ICP35, are components of immature capsids. Here we report that ICP35, ICP5, and UL38 (VP19c) coalesce at late times postinfection and form antigenically dense structures located at the periphery of nuclei, close to but not abutting nuclear membranes. These structures were formed in cells infected with a virus carrying a temperature-sensitive mutation in the UL15 gene at nonpermissive temperatures. Since at these temperatures viral DNA is made but not packaged, these structures must contain the proteins for immature-capsid assembly and were therefore designated assemblons. These assemblons are located at the periphery of a diffuse structure composed of proteins involved in DNA synthesis. This structure overlaps only minimally with the assemblons. In contrast, tegument proteins were located in asymmetrically distributed structures also partially overlapping with assemblons but frequently located nearer to nuclear membranes. Of particular interest is the finding that the UL15 protein colocalized with the proteins associated with viral DNA synthesis rather than with assemblons, suggesting that the association with DNA may take place during its synthesis and precedes the involvement of this protein in packaging of the viral DNA into capsids. The formation of three different compartments consisting of proteins involved in viral DNA synthesis, the capsid proteins, and tegument proteins suggests that there exists a viral machinery which enables aggregation and coalescence of specific viral protein groups on the basis of their function. PMID:8676489

Ward, P L; Ogle, W O; Roizman, B

1996-01-01

237

Virus-Induced Chaperone-Enriched (VICE) Domains Function as Nuclear Protein Quality Control Centers during HSV1 Infection  

Microsoft Academic Search

Virus-Induced Chaperone-Enriched (VICE) domains form adjacent to nuclear viral replication compartments (RC) during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70), the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains.

Christine M. Livingston; Marius F. Ifrim; Ann E. Cowan; Sandra K. Weller

2009-01-01

238

Ubiquitin-Regulated Nuclear-Cytoplasmic Trafficking of the Nipah Virus Matrix Protein Is Important for Viral Budding  

PubMed Central

Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an “off-the-shelf” therapeutic against acute NiV infection. PMID:21085610

Wang, Yao E.; Park, Arnold; Lake, Michael; Pentecost, Mickey; Torres, Betsabe; Yun, Tatyana E.; Wolf, Mike C.; Holbrook, Michael R.

2010-01-01

239

C++ OPPS, a new software for the interpretation of protein dynamics from nuclear magnetic resonance measurements  

NASA Astrophysics Data System (ADS)

Nuclear magnetic resonance (NMR) is a powerful tool for elucidating protein dynamics because of the possibility to interpret nuclear spin relaxation properties in terms of microdynamic parameters. Magnetic relaxation times T1, T2, and NOE depend on dipolar and quadrupolar interactions, on chemical shift anisotropy and cross-correlation effects. Within the framework of given motional model, it is possible to express the NMR relaxation times as functions of spectral densities (Abragam, The Principles of Nuclear Magnetism; Oxford University Press: Clarendon, London, 1961), obtaining the connection between macroscopic observables and microscopic properties. In this context, recently Meirovitch et al. (Shapiro et al., Biochemistry 2002, 41, 6271, Meirovitch et al., J Phys Chem B 2006, 110, 20615, Meirovitch et al., J Phys Chem B 2007, 111, 12865) applied the dynamical model introduced by Polimeno and Freed (Polimeno and Freed, Adv Chem Phys 1993, 83, 89, Polimeno and Freed, J Phys Chem 1995, 99, 10995), known as the slowly relaxing local structure (SRLS) model, to the study of NMR data. The program C++OPPS (http://www.chimica.unipd.it/licc/), developed in our laboratory, implements the SRLS model in an user-friendly way with a graphical user interface (GUI), introduced to simplify the work to users who do not feel at ease with the complex mathematics of the model and the difficulties of command line based programs. The program is an evolution of the old FORTRAN 77 implementation COPPS (COupled Protein Probe Smoluchowski) and presents a number of new features: the presence of an easy to use GUI written in JAVA; high calculation performance thanks to features of C++ language, employment of BLAS (basic linear algebra subprograms) library (Blackford et al., Trans Math Soft 2002, 28, 135) in handling matrix-vector operations and parallelization of the code under the MPI (message passing interface) paradigm (Gropp et al., Parallel Comput 1996, 22, 789, Gropp and Lusk, User's Guide for mpich, a Portable Implementation of MPI Mathematics and Computer Science Division; Argonne National Laboratory, 1996); possibility to predict the diffusion tensor of the protein via a hydrodynamic approach (Barone et al., J Comp Chem, in press). A cluster version of C++OPPS was also developed, which can be easily accessed by users via the web.

Zerbetto, Mirco; Polimeno, Antonino; Meirovitch, Eva

240

The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins.  

PubMed

Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein. We identified several mSufu variants of which some were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. We confirmed the mSufu-SAP18 and mSufu-Galectin3 interactions by independent biochemical assays. Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus. PMID:14611647

Paces-Fessy, Mélanie; Boucher, Dominique; Petit, Emile; Paute-Briand, Sandrine; Blanchet-Tournier, Marie-Françoise

2004-03-01

241

Protein Kinase A Activation Enhances ?-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies  

PubMed Central

The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of ?-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of ?-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on ?-catenin were required for nuclear re-localization. Further, ?-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between ?-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/?-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of ?-catenin and its differential protein-protein interaction that can be modulated by PKA signaling. PMID:25299576

Zhang, Mei; Mahoney, Emilia; Zuo, Tao; Manchanda, Parmeet K.; Davuluri, Ramana V.; Kirschner, Lawrence S.

2014-01-01

242

On the Problem of Establishing the Subcellular Localization of Dictyostelium Retrotransposon TRE5-A Proteins by Biochemical Analysis of Nuclear Extracts  

Microsoft Academic Search

At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein in vivo. Although this assumption will hold true for most of the analyzed proteins, it could also lead to false interpretations. We analyzed the subcellular distribution of endogenous and plasmid-borne proteins derived from the

Ulrich Hentschel; Ilse Zündorf; Theodor Dingermann; Thomas Winckler

2001-01-01

243

Binding of an endosperm-specific nuclear protein to a maize beta-zein gene correlates with zein transcriptional activity  

Microsoft Academic Search

Promoter regions of alpha- and beta-zein genes were analyzed for binding of nuclear proteins from developing endosperm and seedling tissue of maize. Using a band-shift assay, we identified two distinct protein factors, alpha-1 and beta-1, that interacted specifically with alpha- and beta-zein gene promoter regions, respectively. Alpha-1 was present in nuclei from both endosperm and seedling tissue, whereas beta-1 was

Jae-Seong So; Brian A. Larkins

1991-01-01

244

Heat Shock Protein 70 Is Related to Thermal Inhibition of Nuclear Export of the Influenza Virus Ribonucleoprotein Complex  

PubMed Central

The influenza virus genome replicates and forms a viral ribonucleoprotein complex (vRNP) with nucleoprotein (NP) and RNA polymerases in the nuclei of host cells. vRNP is then exported into the cytoplasm for viral morphogenesis at the cell membrane. Matrix protein 1 (M1) and nonstructural protein 2/nuclear export protein (NS2/NEP) work in the nuclear export of vRNP by associating with it. It was previously reported that influenza virus production was inhibited in Madin-Darby canine kidney (MDCK) cells cultured at 41°C because nuclear export of vRNP was blocked by the dissociation of M1 from vRNP (A. Sakaguchi, E. Hirayama, A. Hiraki, Y. Ishida, and J. Kim, Virology 306:244-253, 2003). Previous data also suggested that a certain protein(s) synthesized only at 41°C inhibited the association of M1 with vRNP. The potential of heat shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37°C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1. Moreover, NS2 accumulated in the nucleoli of host cells cultured at 41°C, showing that the export of NS2 was also disturbed at 41°C. However, NS2 was exported normally from the nucleus, irrespective of PGA1 treatment at 37°C, suggesting that HSP70 does not influence NS2. PMID:14722281

Hirayama, Etsuko; Atagi, Hiromitsu; Hiraki, Akihiro; Kim, Jeman

2004-01-01

245

Characterization of nucleotide sequences that interact with a nuclear protein fraction in rRNA gene of Vicia faba  

Microsoft Academic Search

A certain nucleotide sequence in the promoter region of Vicia faba rRNA genes that specifically binds to a nuclear protein fraction has been identified by using a gel retardation assay and DNase I footprinting technique. The binding site of this protein fraction is located about 60 bp upstream from the initiation site of the pre-rRNA transcript. This location does not

Tadaka Nakajima; Akihiro Suzuki; Shigeyuki Tanifuji; Atsushi Kato

1992-01-01

246

A Novel Bipartite Nuclear Localization Signal Guides BPM1 Protein to Nucleolus Suggesting Its Cullin3 Independent Function  

PubMed Central

BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin–proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed. PMID:23251450

Leljak Levanic, Dunja; Horvat, Tomislav; Martincic, Jelena; Bauer, Natasa

2012-01-01

247

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells  

PubMed Central

CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation. PMID:23541585

Eto, Masumi; Kirkbride, Jason A; Chugh, Rishika; Karikari, Nana Kofi; Kim, Jee In

2013-01-01

248

The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.  

PubMed Central

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport. Images PMID:1848177

Rihs, H P; Jans, D A; Fan, H; Peters, R

1991-01-01

249

Nicotine mediates hypochlorous acid-induced nuclear protein damage in mammalian cells.  

PubMed

Activated neutrophils secrete hypochlorous acid (HOCl) into the extracellular space of inflamed tissues. Because of short diffusion distance in biological fluids, HOCl-damaging effect is restricted to the extracellular compartment. The current study aimed at investigating the ability of nicotine, a component of tobacco and electronic cigarettes, to mediate HOCl-induced intracellular damage. We report, for the first time, that HOCl reacts with nicotine to produce nicotine chloramine (Nic-Cl). Nic-Cl caused dose-dependent damage to proliferating cell nuclear antigen (PCNA), a nuclear protein, in cultured mammalian lung and kidney cells. Vitamin C, vitamin E analogue (Trolox), glutathione, and N-acetyl-L-cysteine inhibited the Nic-Cl-induced PCNA damage, implicating oxidation in PCNA damage. These findings point out the ability of nicotine to mediate HOCl-induced intracellular damage and suggest antioxidants as protective measures. The results also raise the possibility that Nic-Cl can be created in the inflamed tissues of tobacco and electronic cigarette smokers and may contribute to smoking-related diseases. PMID:24357417

Salama, Samir A; Arab, Hany H; Omar, Hany A; Maghrabi, Ibrahim A; Snapka, Robert M

2014-06-01

250

Recognition of Transcription Termination Signal by the Nuclear Polyadenylated RNA-binding (NAB) 3 Protein*  

PubMed Central

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the ?-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination. PMID:21084293

Hobor, Fruzsina; Pergoli, Roberto; Kubicek, Karel; Hrossova, Dominika; Bacikova, Veronika; Zimmermann, Michal; Pasulka, Josef; Hofr, Ctirad; Vanacova, Stepanka; Stefl, Richard

2011-01-01

251

Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution  

SciTech Connect

Research highlights: {yields} Verifying by direct methylation assay the substrate sites of PRMT1 in the hnRNP K protein. {yields} Identifying the preferred PMRT1 methylation regions in hnRNP K by kinetic analysis. {yields} Linking methylation in regulating nuclear localization of hnRNP K. -- Abstract: Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.

Chang, Yuan-I; Hsu, Sheng-Chieh [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China)] [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Chau, Gar-Yang [Department of Surgery, Taipei Veterans General Hospital, Taipei 112, Taiwan (China) [Department of Surgery, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Department of Surgery, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan (China); Huang, Chi-Ying F. [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China) [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan (China); Sung, Jung-Sung [Taipei City Hospital, Yang-Ming Branch, Taipei 111, Taiwan (China)] [Taipei City Hospital, Yang-Ming Branch, Taipei 111, Taiwan (China); Hua, Wei-Kai [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China)] [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Lin, Wey-Jinq, E-mail: wjlin@ym.edu.tw [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China) [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Department of Education and Research, Taipei City Hospital, Taipei 103, Taiwan (China)

2011-01-21

252

Different Sets of ER-Resident J-Proteins Regulate Distinct Polar Nuclear-Membrane Fusion Events in Arabidopsis thaliana.  

PubMed

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. PMID:25231968

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-Ichi

2014-11-01

253

Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis  

PubMed Central

Background The Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB. PMID:16008839

Xu, Hongzhi; Somers, Z Brad; Robinson, Melvin L; Hebert, Michael D

2005-01-01

254

The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana  

PubMed Central

During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from ?-Tubulin Complexes (?-TuCs) located at the surface of the nucleus. The molecular mechanisms of ?-TuC association to the nuclear envelope (NE) are currently unknown. The ?-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest ?-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active ?-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of ?-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a ?-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum. PMID:24348487

Batzenschlager, Morgane; Masoud, Kinda; Janski, Natacha; Houlne, Guy; Herzog, Etienne; Evrard, Jean-Luc; Baumberger, Nicolas; Erhardt, Mathieu; Nomine, Yves; Kieffer, Bruno; Schmit, Anne-Catherine; Chaboute, Marie-Edith

2013-01-01

255

Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA).  

PubMed

Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis. PMID:24309115

Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa; Yoon, Hyun-Joo; Yoo, Hae Yong; Choi, Cheol Yong

2014-01-01

256

Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation  

PubMed Central

Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-?2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network. PMID:24573109

Hinde, Elizabeth; Yokomori, Kyoko; Gaus, Katharina; Hahn, Klaus M.; Gratton, Enrico

2014-01-01

257

Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation  

NASA Astrophysics Data System (ADS)

Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-?2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network.

Hinde, Elizabeth; Yokomori, Kyoko; Gaus, Katharina; Hahn, Klaus M.; Gratton, Enrico

2014-02-01

258

Random Screening for Dominant-Negative Mutants of the Cytomegalovirus Nuclear Egress Protein M50?  

PubMed Central

Inactivation of gene products by dominant-negative (DN) mutants is a powerful tool to assign functions to proteins. Here, we present a two-step procedure to establish a random screen for DN alleles, using the essential murine cytomegalovirus gene M50 as an example. First, loss-of-function mutants from a linker-scanning library were tested for inhibition of virus reconstitution with the help of FLP-mediated ectopic insertion of the mutants into the viral genome. Second, DN candidates were confirmed by conditional expression of the inhibitory proteins in the virus context. This allowed the quantification of the inhibitory effect, the identification of the morphogenesis block, and the construction of DN mutants with improved activity. Based on these observations a DN mutant of the homologous gene (UL50) in human cytomegalovirus was predicted and constructed. Our data suggest that a proline-rich sequence motif in the variable region of M50/UL50 represents a new functional site which is essential for nuclear egress of cytomegalovirus capsids. PMID:17376929

Rupp, Brigitte; Ruzsics, Zsolt; Buser, Christopher; Adler, Barbara; Walther, Paul; Koszinowski, Ulrich H.

2007-01-01

259

SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family.  

PubMed

The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. We have cloned the wild-type SMC1 gene. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino-terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1-2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division. PMID:8276886

Strunnikov, A V; Larionov, V L; Koshland, D

1993-12-01

260

Multiple Protein Domains Contribute to Nuclear Import and Cell Toxicity of DUX4, a Candidate Pathogenic Protein for Facioscapulohumeral Muscular Dystrophy  

PubMed Central

DUX4 (Double Homeobox Protein 4) is a nuclear transcription factor encoded at each D4Z4 unit of a tandem-repeat array at human chromosome 4q35. DUX4 constitutes a major candidate pathogenic protein for facioscapulohumeral muscular dystrophy (FSHD), the third most common form of inherited myopathy. A low-level expression of DUX4 compromises cell differentiation in myoblasts and its overexpression induces apoptosis in cultured cells and living organisms. In this work we explore potential molecular determinants of DUX4 mediating nuclear import and cell toxicity. Deletion of the hypothetical monopartite nuclear localization sequences RRRR23, RRKR98 and RRAR148 (i.e. NLS1, NLS2 and NLS3, respectively) only partially delocalizes DUX4 from the cell nuclei. Nuclear entrance guided by NLS1, NLS2 and NLS3 does not follow the classical nuclear import pathway mediated by ?/? importins. NLS and homeodomain mutants from DUX4 are dramatically less cell-toxic than the wild type molecule, independently of their subcellular localization. A triple ?NLS1-2-3 deletion mutant is still partially localized in the nuclei, indicating that additional sequences in DUX4 contribute to nuclear import. Deletion of ?111 amino acids from the C-terminal of DUX4, on a ?NLS1-2-3 background, almost completely re-localizes DUX4 to the cytoplasm, indicating that the C-ter tail contributes to subcellular trafficking of DUX4. Also, C-terminal deletion mutants of DUX4 on a NLS wild type background are less toxic than wild type DUX4. Results reported here indicate that DUX4 possesses redundant mechanisms to assure nuclear entrance and that its various transcription-factor associated domains play an essential role in cell toxicity. PMID:24116060

Corona, Edgardo Daniel; Jacquelin, Daniela; Gatica, Laura; Rosa, Alberto Luis

2013-01-01

261

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells  

SciTech Connect

Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States)] [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States)] [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States) [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

2013-04-26

262

Sde2: A novel nuclear protein essential for telomeric silencing and genomic stability in Schizosaccharomyces pombe  

SciTech Connect

Research highlights: {yields} Sde2 is essential for telomere silencing. {yields} Sde2 is involved in the maintenance of genomic stability. {yields} Sde2 promotes the recruitment of SHREC, a histone deacetylase complex, to telomeres. -- Abstract: Telomeres, specialized domains assembled at the ends of linear chromosomes, are essential for genomic stability in eukaryotes. The formation and maintenance of telomeres are governed by numerous factors such as telomeric repeats, telomere-binding proteins, heterochromatin proteins, and telomerase. Here, we report Sde2, a novel nuclear protein essential for telomeric silencing and genomic stability in the fission yeast Schizosaccharomyces pombe. A deficiency in sde2 results in the derepression of the ura4{sup +} gene inserted near telomeric repeats, and the noncoding transcripts from telomeric regions accumulate in sde2{Delta} cells. The loss of Sde2 function compromises transcriptional silencing at telomeres, and this silencing defect is accompanied by increased levels of acetylated histone H3K14 and RNA polymerase II occupancy at telomeres as well as reduced recruitment of the SNF2 ATPase/histone deacetylase-containing complex SHREC to telomeres. Deletion of sde2 also leads to a higher frequency of mitotic minichromosome loss, and sde2{Delta} cells often form asci that contain spores in abnormal numbers, shapes, or both. In addition, sde2{Delta} cells are highly sensitive to several stresses, including high/low temperatures, bleomycin, which induces DNA damage, and thiabendazole, a microtubule-destabilizing agent. Furthermore, Sde2 genetically interacts with the telomere regulators Taz1, Pof3, and Ccq1. These findings demonstrate that Sde2 cooperates with other telomere regulators to maintain functional telomeres, thereby preventing genomic instability.

Sugioka-Sugiyama, Rie [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan) [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Sugiyama, Tomoyasu, E-mail: sugiyamt@biol.tsukuba.ac.jp [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan) [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012 (Japan)

2011-03-18

263

Human T-cell activation by 14- and 18-kilodalton nuclear proteins of Leishmania infantum.  

PubMed Central

Leishmanial antigens which stimulate T lymphocytes from primed individuals may be candidates for a vaccine. We recently found a significant concordance between the humoral response specific for two proteins from Leishmania infantum promastigotes, p14 and p18, and a positive leishmanin delayed-type hypersensitivity reaction, testifying to the occurrence of cell-mediated immunity. In this communication, we describe a partial characterization of these antigens and an in vitro analysis of their capacity to activate primed human T cells. We showed, by immunofluorescent staining and through analysis of subcellular fractions by Western immunoblotting, that in stationary-phase promastigotes, p14 and p18 were located only in the parasite nuclei; in the middle of the log phase, a transitory and only weak expression outside the nucleus was detected. We then showed that p14 and p18 antigens shared a common epitope(s). Finally, we analyzed the in vitro proliferation and interleukin-2 production induced by leishmanial proteins in human peripheral blood mononuclear cells from sensitized subjects. We showed that in some individuals who have been exposed to L. infantum the specific response to the whole lysate was mostly due to the nuclear antigens. We demonstrated directly the capacity of nitrocellulose-bound p14 and p18 to activate in vitro all of the tested primed peripheral blood mononuclear cells, which contrasted with a lack of stimulatory activity of other membrane-bound leishmanial proteins. Taken together, our results suggest that an antigenic determinant(s) dominant for some individuals might exist on both antigens. PMID:7558278

Suffia, I; Quaranta, J F; Eulalio, M C; Ferrua, B; Marty, P; Le Fichoux, Y; Kubar, J

1995-01-01

264

ATM Protein Physically and Functionally Interacts with Proliferating Cell Nuclear Antigen to Regulate DNA Synthesis*  

PubMed Central

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase ?, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner. PMID:22362778

Gamper, Armin M.; Choi, Serah; Matsumoto, Yoshihiro; Banerjee, Dibyendu; Tomkinson, Alan E.; Bakkenist, Christopher J.

2012-01-01

265

A new nuclear suppressor system for a mitochondrial RNA polymerase mutant identifies an unusual zinc-finger protein and a polyglutamine domain protein in Saccharomyces cerevisiae.  

PubMed

A yeast strain with a point mutation in the nuclear gene for the core subunit of mitochondrial RNA polymerase was used to isolate new extragenic suppressors. Spontaneously occurring phenotypical revertants were analysed by crosses with the wild-type and tetrad dissection. One of the new nuclear suppressor mutants was characterized by temperature-sensitive growth on non-fermentable carbon sources. This mutant was transformed with a genomic yeast library. Two independent types of DNA clones were isolated which both complemented the temperature-sensitive defect. Subcloning and DNA sequencing identified two novel yeast genes which code for proteins with the characteristic features of transcription factors. Both factors exhibit highly structured protein domains consisting of runs and clusters of asparagine and glutamine residues. One of the proteins contains in addition zinc-finger domains of the C2H2-type. Therefore the genes are proposed to be named AZF1 (asparagine-rich zinc-finger protein) and PGD1 (polyglutamine domain protein). Gene disruption of both reading frames has no detectable influence on the vegetative growth on complete glucose or glycerol media, indicating that the genes may act as high copy number suppressors of the mutant defect. Additional transformation experiments showed that AZF1 is also an efficient suppressor for the original defect in the core subunit of mitochondrial RNA polymerase. PMID:7975891

Bröhl, S; Lisowsky, T; Riemen, G; Michaelis, G

1994-06-01

266

Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.  

PubMed Central

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8637884

Zupan, J R; Citovsky, V; Zambryski, P

1996-01-01

267

The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light-dependent post-  

E-print Network

The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light Charles E. Young Dr. South, Los Angeles, CA 90095-1606, USA Summary Cryptochrome 2 is a ¯avin-type blue light receptor mediating ¯oral induction in response to photoperiod and a blue light-induced hypocotyl

Lin, Chentao

268

Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA.  

PubMed Central

The influenza virus NS1 protein is the only known example of a protein that inhibits the nuclear export of mRNA. To identify the functional domains of this protein, we introduced 18 2- or 3-amino-acid substitutions at approximately equally spaced locations along the entire length of the protein. Two functional domains were identified. The domain near the amino end (amino acids 19 through 38) was shown to be the RNA-binding domain, by using a gel shift assay with purified NS1 protein and spliced viral NS2 mRNA as the RNA target. The second domain, which is in the carboxy half of the molecule, was presumed to be the effector domain that interacts with host nuclear proteins to carry out the nuclear RNA export function, by analogy with the effector domain of the Rev proteins of human immunodeficiency virus (HIV) and other lentiviruses which facilitate rather than inhibit nuclear RNA export. The NS1 protein has a 10-amino-acid sequence that is similar to the consensus sequence in the effector domains of lentivirus Rev proteins, specifically including two crucial leucines at positions 7 and 9 of this sequence. However, the effector domains of the NS1 and Rev (HIV type 1 [HIV-1]) proteins differed in several significant ways including the following: (i) unlike the HIV-1 Rev protein, NS1 effector domain mutants were negative recessive rather than negative dominant, (ii) the NS1 effector domain is about three times larger than the effector domain of the HIV-1 Rev protein, and (iii) unlike the HIV-1 protein, NS1 effector domain mutants exhibited a surprising property, a changed intracellular/intranuclear distribution, compared with the wild-type protein. These differences strongly suggest that the effector domains of the NS1 and Rev proteins interact with different nuclear protein targets, which likely explains the opposite effects of these two proteins on nuclear mRNA export. Images PMID:8139028

Qian, X Y; Alonso-Caplen, F; Krug, R M

1994-01-01

269

Protein interactions at the higher plant nuclear envelope: evidence for a linker of nucleoskeleton and cytoskeleton complex  

PubMed Central

Following the description of SAD1/UNC84 (SUN) domain proteins in higher plants, evidence has rapidly increased that plants contain a functional linker of nucleoskeleton and cytoskeleton (LINC) complex bridging the nuclear envelope (NE). While the SUN domain proteins appear to be highly conserved across kingdoms, other elements of the complex are not and some key components and interactions remain to be identified. This mini review examines components of the LINC complex, including proteins of the SUN domain family and recently identified plant Klarsicht/Anc/Syne-1 homology (KASH) domain proteins. First of these to be described were WIPs (WPP domain interacting proteins), which act as protein anchors in the outer NE. The plant KASH homologs are C-terminally anchored membrane proteins with the extreme C-terminus located in the nuclear periplasm; AtWIPs contain a highly conserved X-VPT motif at the C-terminus in contrast to PPPX in opisthokonts. The role of the LINC complex in organisms with a cell wall, and description of further LINC complex components will be considered, together with other potential plant-specific functions. PMID:24847341

Evans, David E.; Pawar, Vidya; Smith, Sarah J.; Graumann, Katja

2014-01-01

270

Nuclear export of the influenza virus ribonucleoprotein complex: Interaction of Hsc70 with viral proteins M1 and NS2  

PubMed Central

The influenza virus replicates in the host cell nucleus, and the progeny viral ribonucleoprotein complex (vRNP) is exported to the cytoplasm prior to maturation. NS2 has a nuclear export signal that mediates the nuclear export of vRNP by the vRNP–M1–NS2 complex. We previously reported that the heat shock cognate 70 (Hsc70) protein binds to M1 protein and mediates vRNP export. However, the interactions among M1, NS2, and Hsc70 are poorly understood. In the present study, we demonstrate that Hsc70 interacts with M1 more strongly than with NS2 and competes with NS2 for M1 binding, suggesting an important role of Hsc70 in the nuclear export of vRNP. PMID:25161876

Watanabe, Ken; Shimizu, Teppei; Noda, Saiko; Tsukahara, Fujiko; Maru, Yoshiro; Kobayashi, Nobuyuki

2014-01-01

271

Arginine methylation of yeast mRNA-binding protein Npl3 directly affects its function, nuclear export, and intranuclear protein interactions.  

PubMed

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels of methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmt1. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmt1, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins. PMID:15998636

McBride, Anne E; Cook, Jeffrey T; Stemmler, Elizabeth A; Rutledge, Kate L; McGrath, Kelly A; Rubens, Jeffrey A

2005-09-01

272

Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo.  

PubMed Central

We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin. Images PMID:1849072

Prendergast, G C; Ziff, E B

1991-01-01

273

The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome  

PubMed Central

The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger ribonucleoproteins (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance. PMID:24152732

Niepel, Mario; Molloy, Kelly R.; Williams, Rosemary; Farr, Julia C.; Meinema, Anne C.; Vecchietti, Nicholas; Cristea, Ileana M.; Chait, Brian T.; Rout, Michael P.; Strambio-De-Castillia, Caterina

2013-01-01

274

An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region  

PubMed Central

We have identified an integral membrane protein of 145 kD (estimated by SDS-PAGE) of rat liver nuclear envelopes that binds to WGA. We obtained peptide sequence from purified p145 and cloned and sequenced several cDNA clones and one genomic clone. The relative molecular mass of p145 calculated from its complete, cDNA deduced primary structure is 120.7 kD. Antibodies raised against a synthetic peptide represented in p145 reacted monospecifically with p145. In indirect immunofluorescence these antibodies gave punctate staining of the nuclear envelope. Immunogold EM showed specific decoration of the nuclear pores. Thus p145 is an integral membrane protein located specifically in the "pore membrane" domain of the nuclear envelope. To indicate this specific location, and based on its calculated relative molecular mass, the protein is termed POM 121 (pore membrane protein of 121 kD). The 1,199- residue-long primary structure shows a hydrophobic region (residues 29- 72) that is likely to form one (or two adjacent) transmembrane segment(s). The bulk of the protein (residues 73-1199) is predicted to be exposed not on the cisternal side but on the pore side of the pore membrane. It contains 36 consensus sites for various kinases. However, its most striking feature is a repetitive pentapeptide motif XFXFG that has also been shown to occur in several nucleoporins. This nucleoporin- like domain of POM 121 is proposed to function in anchoring components of the nuclear pore complex to the pore membrane. PMID:8335683

1993-01-01

275

The Angelman Syndrome-Associated Protein, E6-AP, Is a Coactivator for the Nuclear Hormone Receptor Superfamily  

PubMed Central

In this study, we found that the E6-associated protein (E6-AP/UBE3A) directly interacts with and coactivates the transcriptional activity of the human progesterone receptor (PR) in a hormone-dependent manner. E6-AP also coactivates the hormone-dependent transcriptional activities of the other members of the nuclear hormone receptor superfamily. Previously, it was shown that E6-AP serves the role of a ubiquitin-protein ligase (E3) in the presence of the E6 protein from human papillomavirus types 16 and 18. Our data show that the ubiquitin-protein ligase function of E6-AP is dispensable for its ability to coactivate nuclear hormone receptors, showing that E6-AP possesses two separable independent functions, as both a coactivator and a ubiquitin-protein ligase. Disruption of the maternal copy of E6-AP is correlated with Angelman syndrome (AS), a genetic neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms. However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. To correlate the E6-AP coactivator function and ubiquitin-protein ligase functions with the AS phenotype, we expressed mutant forms of E6-AP isolated from AS patients and assessed the ability of each of these mutant proteins to coactivate PR or provide ubiquitin-protein ligase activity. This analysis revealed that in the majority of the AS patients examined, the ubiquitin-protein ligase function of E6-AP was defective whereas the coactivator function was intact. This finding suggests that the AS phenotype results from a defect in the ubiquitin-proteosome protein degradation pathway. PMID:9891052

Nawaz, Zafar; Lonard, David M.; Smith, Carolyn L.; Lev-Lehman, Efrat; Tsai, Sophia Y.; Tsai, Ming-Jer; O'Malley, Bert W.

1999-01-01

276

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.  

PubMed Central

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7761467

Azuma, Y; Tabb, M M; Vu, L; Nomura, M

1995-01-01

277

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana  

NASA Technical Reports Server (NTRS)

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

1992-01-01

278

B-lymphoblastic leukemia/lymphoma: overexpression of nuclear DNA repair protein PARP-1 correlates with antiapoptotic protein Bcl-2 and complex chromosomal abnormalities.  

PubMed

Poly(ADP-ribose) polymerase-1 (PARP-1) and Bcl-2 are emerging as therapeutic targets in various cancers. The former is a DNA repair protein associated with genomic stability and apoptosis, whereas the latter is an antiapoptotic protein having a DNA repair function through inhibition of PARP-1. Because genomic stability is critical for prognosis in B-lymphoblastic leukemia/lymphoma (B-ALL), we studied the expression of PARP-1 and Bcl-2 proteins in patients with B-ALL of different ages and compared the results with cytogenetic data. The PARP-1 protein was overexpressed in about two-thirds (61%) of patients with B-ALL. It had a nuclear location, whereas Bcl-2 protein was cytosolic. Expression of the 2 proteins showed a highly positive correlation (? = 0.367; P < .001). Overexpression of PARP-1 correlated with a complex karyotype (P = .030), and this correlation remained significant for coexpression of PARP-1 and Bcl-2 proteins (?(2) = 7.498; P = .024) as well as after exclusion of pediatric patients (n = 9, P = .042). Overexpression of PARP-1 was not significantly more common in diploid versus aneuploid karyotypes (50% versus 59%, P = .610). The PARP-1 protein showed no correlation with specific chromosomal abnormalities associated with prognosis in B-ALL, as defined by the World Health Organization. In conclusion, high expression of the PARP-1 protein among patients with B-ALL is related to a complex karyotype and Bcl-2 positivity. Although these findings require validation in a larger population, the observations will be valuable in planning therapeutic trials (such as of PARP inhibitors and BH3 mimetics). PMID:24856976

Pournazari, Payam; Padmore, Ruth F; Kosari, Farid; Scalia, Peter; Shahbani-Rad, Meer-Taher; Shariff, Sami; Demetrick, Douglas J; Bosch, Mark; Mansoor, Adnan

2014-08-01

279

Nuclear LSm8 affects number of cytoplasmic processing bodies via controlling cellular distribution of Like-Sm proteins  

PubMed Central

Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2–7 proteins. We propose a model in which redistribution of LSm2–7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies. PMID:22875987

Novotny, Ivan; Podolska, Katerina; Blazikova, Michaela; Valasek, Leos Shivaya; Svoboda, Petr; Stanek, David

2012-01-01

280

MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells.  

PubMed

Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle. PMID:24950247

Jafferali, Mohammed Hakim; Vijayaraghavan, Balaje; Figueroa, Ricardo A; Crafoord, Ellinor; Gudise, Santhosh; Larsson, Veronica J; Hallberg, Einar

2014-10-01

281

Adenoviral E1B-55kDa protein inhibits yeast mRNA export and perturbs nuclear structure.  

PubMed Central

The mechanisms of export of RNA from the nucleus are poorly understood; however, several viral proteins modulate nucleocytoplasmic transport of mRNA. Among these are the adenoviral proteins E1B-55kDa and E4-34kDa. Late in infection, these proteins inhibit export of host transcripts and promote export of viral mRNA. To investigate the mechanism by which these proteins act, we have expressed them in Saccharomyces cerevisiae. Overexpression of either or both proteins has no obvious effect on cell growth. By contrast, overexpression of E1B-55kDa bearing a nuclear localization signal (NLS) dramatically inhibits cell growth. In this situation, the NLS-E1B-55kDa protein is localized to the nuclear periphery, fibrous material is seen in the nucleoplasm, and poly(A)+ RNA accumulates in the nucleus. Simultaneous overexpression of E4-34kDa bearing or lacking an NLS does not modify these effects. We discuss the mechanisms of selective mRNA transport. Images Fig. 1 Fig. 2 Fig. 3 PMID:7638199

Liang, S; Hitomi, M; Tartakoff, A M

1995-01-01

282

Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function  

SciTech Connect

We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

Dement, Gregory A. [School of Molecular Biosciences, Washington State University, Rm. 639, Fulmer Hall, Pullman, WA 99164-4660 (United States); Maloney, Scott C. [School of Molecular Biosciences, Washington State University, Rm. 639, Fulmer Hall, Pullman, WA 99164-4660 (United States); Reeves, Raymond [School of Molecular Biosciences, Washington State University, Rm. 639, Fulmer Hall, Pullman, WA 99164-4660 (United States)]. E-mail: reevesr@mail.wsu.edu

2007-01-01

283

X-linked adrenal hypoplasia congenita is caused by abnormal nuclear localization of the DAX-1 protein  

PubMed Central

Mutations in the DAX-1 [dosage-sensitive sex reversal–adrenal hypoplasia congenita (AHC) critical region on the X chromosome; NR0B1] gene cause X-linked AHC associated with hypogonadotropic hypogonadism. DAX-1 encodes an unusual orphan member of the nuclear hormone receptor superfamily, acting as a transcriptional repressor of genes involved in the steroidogenic pathway. All DAX-1 mutations found in AHC patients alter the protein C terminus, which shares similarity to the ligand binding domain of nuclear hormone receptors and bears transcriptional repressor activity. This property is invariably impaired in DAX-1 AHC mutants. Here we show that the localization of DAX-1 AHC mutant proteins is drastically shifted toward the cytoplasm, even if their nuclear localization signal, which resides in the N terminal of the protein, is intact. Cytoplasmic localization of DAX-1 AHC mutants correlates with an impairment in their transcriptional repression activity. These results reveal a critical role of an intact C terminus in determining DAX-1 subcellular localization and constitute an important example of a defect in human organogenesis caused by impaired nuclear localization of a transcription factor. PMID:12034880

Lehmann, Sylvia G.; Lalli, Enzo; Sassone-Corsi, Paolo

2002-01-01

284

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging  

SciTech Connect

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.

You, Jae-Hwan; Howell, Gareth [Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds (United Kingdom); Pattnaik, Asit K.; Osorio, Fernando A. [Nebraska Center for Virology, E249A Beadle, Lincoln (United States); Hiscox, Julian A. [Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds (United Kingdom); Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds (United Kingdom)], E-mail: j.a.hiscox@leeds.ac.uk

2008-08-15

285

Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization  

PubMed Central

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. PMID:24376827

Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

2013-01-01

286

Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation  

PubMed Central

We present a generally applicable approach for monitoring protein aggregation by detecting changes in surface hydration water dynamics and the changes in solvent accessibility of specific protein sites, as protein aggregation proceeds in solution state. This is made possible through the Overhauser dynamic nuclear polarization (DNP) of water interacting with stable nitroxide spin labels tethered to specific proteins sites. This effect is highly localized due to the magnetic dipolar nature of the electron–proton spin interaction, with >80 % of their interaction occurring within 5 Å between the unpaired electron of the spin label and the proton of water. We showcase our tool on the aggregation of tau proteins, whose fibrillization is linked to neurodegenerative disease pathologies known as taupathies. We demonstrate that the DNP approach to monitor local changes in hydration dynamics with residue specificity and local contrast can distinguish specific and neat protein-protein packing leading to fibers from non-specific protein agglomeration or precipitation. The ability to monitor tau assembly with local, residue-specific, resolution, under ambient condition and in solution state will help unravel the mechanism and structural characteristics of the gradual process of tau aggregation into amyloid fibers, which remains unclear to this day. PMID:19639158

Pavlova, Anna; McCarney, Evan R.; Peterson, Dylan W.; Dahlquist, Frederick W.; Lew, John; Han, Songi

2011-01-01

287

Fibroblast growth factor-223 binds directly to the survival of motoneuron protein and is associated with small nuclear RNAs  

PubMed Central

The SMN (survival of motoneuron) protein is mutated in patients with the neurodegenerative disease spinal muscular atrophy. We have shown previously that a high-molecular-mass isoform of FGF (fibroblast growth factor) 2 (FGF-223) is in a complex with SMN [Claus, Döring, Gringel, Müller-Ostermeyer, Fuhlrott, Kraft and Grothe (2003) J. Biol. Chem. 278, 479–485]. FGF-2 is a neurotrophic factor for motoneurons, and is known not only as a classical extracellular growth factor, but also as a nuclear protein. In the present study, we demonstrate that SMN binds to the arginine-rich N-terminus of FGF-223. In turn, FGF-223 interacts with amino acid residues 1–90 of the human SMN protein. This sequence displays nucleic-acid-binding capacity and overlaps partially with known binding sites for Gemin2/SIP1 (SMN-interacting protein 1) and p53. Finally, as a functional consequence of FGF-223 binding to SMN, FGF-223 is in a complex with the small nuclear RNAs U2 and U4. Since SMN functions as an assembly factor for snRNPs (small nuclear ribonucleoprotein particles), these results suggest binding of FGF-223 to snRNPs. PMID:15222879

2004-01-01

288

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin  

PubMed Central

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance. PMID:19927119

Bancaud, Aurelien; Huet, Sebastien; Daigle, Nathalie; Mozziconacci, Julien; Beaudouin, Joel; Ellenberg, Jan

2009-01-01

289

Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.  

PubMed Central

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins. Images PMID:3110598

Swanson, M S; Nakagawa, T Y; LeVan, K; Dreyfuss, G

1987-01-01

290

Cytoplasmic-nuclear shuttling of the urokinase mRNA binding protein regulates message stability.  

PubMed

Treatment of small airway epithelial (SAEC) cells or lung epithelial (Beas2B) cells with TNF-alpha or PMA induces urokinase-type plasminogen activator (uPA) expression. Treatment of these cells with TNF-alpha, PMA or cycloheximide but not TGF-beta increased steady-state expression of uPAmRNA. TNF-alpha, PMA or cycloheximide caused 8-10 fold extensions of the uPAmRNA half-life in SAEC or Beas2B cells treated with DRB, a transcriptional inhibitor. These findings suggest that uPA gene expression involves a post-transcriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 30 kDa uPA mRNA binding protein (uPA mRNABp) that selectively binds to a 66 nt protein binding fragment of uPA mRNA containing regulatory information for message stabilization. Binding of cytoplasmic uPA mRNABp to uPA mRNA was abolished after treatment with TNF-alpha but not TGF-beta. In addition, we found the accumulation of 30 kDa uPAmRNABp in the nuclear extracts of TNF-alpha but not TGF-beta treated cells. The uPA mRNABp starts moving to the nucleus from the cytoplasmic compartment as early as three hours after TNF-alpha treatment. Complete translocation is achieved between 12-24 h, which coincides with the maximal expression of uPA protein effected by cytokine stimulation. Treatment of Beas2B cells with NaF inhibited TNF-alpha-mediated translocation of uPA mRNABp from the cytoplasm to the nucleus and concomitant inhibition of uPA expression. TNF-alpha stabilizes uPA mRNA by translocating the uPA mRNABp from the cytoplasm to the nucleus. Our results demonstrate a novel mechanism governing uPA mRNA stability through shuttling of uPA mRNABp between the nucleus and cytoplasm. This newly identified pathway may have evolved to regulate uPA-mediated functions of the lung epithelium in inflamation or neoplasia. PMID:12236587

Shetty, Sreerama

2002-08-01

291

Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells  

SciTech Connect

Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies.

Sides, Mark D. [Department of Medicine, Section of Pulmonary Disease and Critical Care, Tulane University School of Medicine, New Orleans, LA (United States); Block, Gregory J. [University of Washington Institute for Stem Cell and Regenerative Medicine, Seattle, WA (United States); Shan, Bin; Esteves, Kyle C. [Department of Medicine, Section of Pulmonary Disease and Critical Care, Tulane University School of Medicine, New Orleans, LA (United States); Lin, Zhen; Flemington, Erik K. [Department of Pathology, Tulane University School of Medicine, New Orleans, LA (United States); Lasky, Joseph A., E-mail: jlasky@tulane.edu [Department of Medicine, Section of Pulmonary Disease and Critical Care, Tulane University School of Medicine, New Orleans, LA (United States)

2011-06-20

292

Identification of Nuclear and Nucleolar Localization Signals of Pseudorabies Virus (PRV) Early Protein UL54 Reveals that Its Nuclear Targeting Is Required for Efficient Production of PRV ?  

PubMed Central

The pseudorabies virus (PRV) early protein UL54 is a homologue of herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein that is essential for HSV-1 infection. In this study, the subcellular localization and nuclear import signals of PRV UL54 were characterized. UL54 was shown to predominantly localize to the nucleolus in transfected cells. By constructing a series of mutants, a functional nuclear localization signal (NLS) and a genuine nucleolar localization signal (NoLS) of UL54 were for the first time identified and mapped to amino acids 61RQRRR65 and 45RRRRGGRGGRAAR57, respectively. Additionally, three recombinant viruses with mutations of the NLS and/or the NoLS in UL54 were constructed based on PRV bacterial artificial chromosome (BAC) pBecker2 to test the effect of UL54 nuclear targeting on viral replication. In comparison with the wild-type virus, a recombinant virus harboring an NLS or NoLS mutation of UL54 reduced viral production to different extents. However, mutations of both the NLS and NoLS targeted UL54 to the cytoplasm in recombinant virus-infected cells and significantly impaired viral replication, comparable to the UL54-null virus. In addition, a virus lacking the NLS or the NoLS displayed modest defects in viral gene expression and DNA synthesis. However, deletion of both the NLS and the NoLS resulted in severe defects in viral gene expression and DNA synthesis, as well as production of infectious progeny. Thus, we have identified a classical NLS and a genuine NoLS in UL54 and demonstrate that the nuclear targeting of UL54 is required for efficient production of PRV. PMID:21795331

Li, Meili; Wang, Shuai; Cai, Mingsheng; Zheng, Chunfu

2011-01-01

293

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein  

SciTech Connect

A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio (Aichi Cancer Center Research Inst., Nagoya (Japan))

1991-10-01

294

Poly(A) binding protein nuclear 1 levels affect alternative polyadenylation.  

PubMed

The choice for a polyadenylation site determines the length of the 3'-untranslated region (3'-UTRs) of an mRNA. Inclusion or exclusion of regulatory sequences in the 3'-UTR may ultimately affect gene expression levels. Poly(A) binding protein nuclear 1 (PABPN1) is involved in polyadenylation of pre-mRNAs. An alanine repeat expansion in PABPN1 (exp-PABPN1) causes oculopharyngeal muscular dystrophy (OPMD). We hypothesized that previously observed disturbed gene expression patterns in OPMD muscles may have been the result of an effect of PABPN1 on alternative polyadenylation, influencing mRNA stability, localization and translation. A single molecule polyadenylation site sequencing method was developed to explore polyadenylation site usage on a genome-wide level in mice overexpressing exp-PABPN1. We identified 2012 transcripts with altered polyadenylation site usage. In the far majority, more proximal alternative polyadenylation sites were used, resulting in shorter 3'-UTRs. 3'-UTR shortening was generally associated with increased expression. Similar changes in polyadenylation site usage were observed after knockdown or overexpression of expanded but not wild-type PABPN1 in cultured myogenic cells. Our data indicate that PABPN1 is important for polyadenylation site selection and that reduced availability of functional PABPN1 in OPMD muscles results in use of alternative polyadenylation sites, leading to large-scale deregulation of gene expression. PMID:22772983

de Klerk, Eleonora; Venema, Andrea; Anvar, S Yahya; Goeman, Jelle J; Hu, Ouhua; Trollet, Capucine; Dickson, George; den Dunnen, Johan T; van der Maarel, Silvère M; Raz, Vered; 't Hoen, Peter A C

2012-10-01

295

Polyalanine-independent Conformational Conversion of Nuclear Poly(A)-binding Protein 1 (PABPN1)*  

PubMed Central

Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation. PMID:22570486

Winter, Reno; Kuhn, Uwe; Hause, Gerd; Schwarz, Elisabeth

2012-01-01

296

Poly(A) binding protein nuclear 1 levels affect alternative polyadenylation  

PubMed Central

The choice for a polyadenylation site determines the length of the 3?-untranslated region (3?-UTRs) of an mRNA. Inclusion or exclusion of regulatory sequences in the 3?-UTR may ultimately affect gene expression levels. Poly(A) binding protein nuclear 1 (PABPN1) is involved in polyadenylation of pre-mRNAs. An alanine repeat expansion in PABPN1 (exp-PABPN1) causes oculopharyngeal muscular dystrophy (OPMD). We hypothesized that previously observed disturbed gene expression patterns in OPMD muscles may have been the result of an effect of PABPN1 on alternative polyadenylation, influencing mRNA stability, localization and translation. A single molecule polyadenylation site sequencing method was developed to explore polyadenylation site usage on a genome-wide level in mice overexpressing exp-PABPN1. We identified 2012 transcripts with altered polyadenylation site usage. In the far majority, more proximal alternative polyadenylation sites were used, resulting in shorter 3?-UTRs. 3?-UTR shortening was generally associated with increased expression. Similar changes in polyadenylation site usage were observed after knockdown or overexpression of expanded but not wild-type PABPN1 in cultured myogenic cells. Our data indicate that PABPN1 is important for polyadenylation site selection and that reduced availability of functional PABPN1 in OPMD muscles results in use of alternative polyadenylation sites, leading to large-scale deregulation of gene expression. PMID:22772983

Venema, Andrea; Anvar, S. Yahya; Goeman, Jelle J.; Hu, OuHua; Trollet, Capucine; Dickson, George; den Dunnen, Johan T.; van der Maarel, Silvere M.; Raz, Vered; 't Hoen, Peter A. C.

2012-01-01

297

Nuclear protein phosphatases with Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis  

PubMed Central

Perception of the plant steroid hormone brassinolide (BL) by the membrane-associated receptor kinase BRI1 triggers the dephosphorylation and accumulation in the nucleus of the transcriptional modulators BES1 and BZR1. We identified bsu1-1D as a dominant suppressor of bri1 in Arabidopsis. BSU1 encodes a nuclear-localized serine–threonine protein phosphatase with an N-terminal Kelch-repeat domain, and is preferentially expressed in elongating cells. BSU1 is able to modulate the phosphorylation state of BES1, counteracting the action of the glycogen synthase kinase-3 BIN2, and leading to increased steady-state levels of dephosphorylated BES1. BSU1 belongs to a small gene family; loss-of-function analyses unravel the extent of functional overlap among members of the family and confirm the role of these phosphatases in the control of cell elongation by BL. Our data indicate that BES1 is subject to antagonistic phosphorylation and dephosphorylation reactions in the nucleus, which fine-tune the amplitude of the response to BL. PMID:14977918

Mora-Garcia, Santiago; Vert, Gregory; Yin, Yanhai; Cano-Delgado, Ana; Cheong, Hyeonsook; Chory, Joanne

2004-01-01

298

Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation  

PubMed Central

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression. PMID:22510880

Wilson, Catherine H; Crombie, Catriona; van der Weyden, Louise; Poulogiannis, George; Rust, Alistair G; Pardo, Mercedes; Gracia, Tannia; Yu, Lu; Choudhary, Jyoti; Poulin, Gino B; McIntyre, Rebecca E; Winton, Douglas J; March, H Nikki; Arends, Mark J; Fraser, Andrew G; Adams, David J

2012-01-01

299

Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins  

SciTech Connect

Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin {alpha} in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin {alpha}, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin {alpha} interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.

Cassell, Geoffrey D. [Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037 (United States); Weitzman, Matthew D. [Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037 (United States)]. E-mail: weitzman@salk.edu

2004-10-01

300

Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2? in the human osteosarcoma cell line U2OS  

PubMed Central

Background Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2? plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2? in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. Results By knocking down the expression of LAP2?, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2? knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2? knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2? knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. Conclusions Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2? reduced expression. Our study implies on a novel role of LAP2? in the maintenance of cell ploidy status. LAP2? depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis. PMID:24472424

2014-01-01

301

Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene  

PubMed Central

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production. PMID:23966414

Copeland, Anna Maria; Altamura, Louis A.; Van Deusen, Nicole M.

2013-01-01

302

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1  

PubMed Central

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. PMID:23938469

Soares, Iaci N.; Caetano, Fabiana A.; Pinder, Jordan; Rodrigues, Bruna Roz; Beraldo, Flavio H.; Ostapchenko, Valeriy G.; Durette, Chantal; Pereira, Grace Schenatto; Lopes, Marilene H.; Queiroz-Hazarbassanov, Nicolle; Cunha, Isabela W.; Sanematsu, Paulo I.; Suzuki, Sergio; Bleggi-Torres, Luiz F.; Schild-Poulter, Caroline; Thibault, Pierre; Dellaire, Graham; Martins, Vilma R.; Prado, Vania F.; Prado, Marco A. M.

2013-01-01

303

Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1.  

PubMed

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. PMID:23938469

Soares, Iaci N; Caetano, Fabiana A; Pinder, Jordan; Rodrigues, Bruna Roz; Beraldo, Flavio H; Ostapchenko, Valeriy G; Durette, Chantal; Pereira, Grace Schenatto; Lopes, Marilene H; Queiroz-Hazarbassanov, Nicolle; Cunha, Isabela W; Sanematsu, Paulo I; Suzuki, Sergio; Bleggi-Torres, Luiz F; Schild-Poulter, Caroline; Thibault, Pierre; Dellaire, Graham; Martins, Vilma R; Prado, Vania F; Prado, Marco A M

2013-11-01

304

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry  

PubMed Central

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A2 domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA2 activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism. PMID:22696661

Popa-Wagner, Ruth; Porwal, Manvi; Kann, Michael; Reuss, Matthias; Weimer, Marc; Florin, Luise

2012-01-01

305

The maize abscisic acid-responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals.  

PubMed Central

The maize abscisic acid (ABA)-responsive rab17 mRNA and Rab17 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rab17 mRNA and Rab17 protein were found in all cells of embryo tissues. Synthesis of rab17 mRNA occurred initially in the embryo axis. As maturation progressed, rab17 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rab17 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rab17 and a nuclear localization signal (NLS) binding phosphoprotein, Nopp140, interaction of Rab17 with NLS peptides was studied. We found specific binding of Rab17 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rab17 was found to be dependent upon phosphorylation. These results suggest that Rab17 may play a role in nuclear protein transport. PMID:8180497

Goday, A; Jensen, A B; Culianez-Macia, F A; Mar Alba, M; Figueras, M; Serratosa, J; Torrent, M; Pages, M

1994-01-01

306

Impact of VP1-specific protein sequence motifs on adeno-associated virus type 2 intracellular trafficking and nuclear entry.  

PubMed

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism. PMID:22696661

Popa-Wagner, Ruth; Porwal, Manvi; Kann, Michael; Reuss, Matthias; Weimer, Marc; Florin, Luise; Kleinschmidt, Jürgen A

2012-09-01

307

Nuclear magnetic resonance signal chemical shifts and molecular simulations: a multidisciplinary approach to modeling copper protein structures.  

PubMed

Nuclear magnetic resonance (NMR) chemical shifts are experimental observables that are available during the first stage of the protein structure determination process. Recently, some methodologies for building structural models of proteins using only these experimental data have been implemented. To assess the potential of these methods for modeling metalloproteins (generally considered a challenging benchmark), we determined the structures of the yeast copper chaperone Atx1 and the CuA domain of Thermus thermophilus cytochrome c oxidase starting from the available chemical shift data. The metal centers were modeled using molecular dynamics simulations with molecular mechanics potentials. The results obtained are evaluated and discussed. PMID:21842229

Sgrignani, Jacopo; Pierattelli, Roberta

2012-01-01

308

Nuclear loss of protein arginine N-methyltransferase 2 in breast carcinoma is associated with tumor grade and overexpression of cyclin D1 protein.  

PubMed

Human protein arginine N-methyltransferase 2 (PRMT2, HRMT1L1) is a protein that belongs to the arginine methyltransferase family, and it has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. In this study, we provide evidences for the negative effect of PRMT2 on breast cancer cell proliferation in vitro and in vivo. Morever, cyclin D1, one of the key modulators of cell cycle, was found to be downregulated by PRMT2, and PRMT2 was further shown to suppress the estrogen receptor ?-binding affinity to the activator protein-1 (AP-1) site in cyclin D1 promoter through indirect binding with AP-1 site, resulting in the inhibition of cyclin D1 promoter activity in MCF-7 cells. Furthermore, a positive correlation between the expression of PRMT2 and cyclin D1 was confirmed in the breast cancer tissues by using tissue microarray assay. In addition, PRMT2 was found to show a high absent percentage in breast caner cell nuclei and the nuclear loss ratio of PRMT2 was demonstrated to positively correlate with cyclin D1 expression and the increasing tumor grade of invasive ductal carcinoma. Those results offer an essential insight into the effect of PRMT2 on breast carcinogenesis, and PRMT2 nuclear loss might be an important biological marker for the diagnosis of breast cancer. PMID:24292672

Zhong, J; Cao, R-X; Liu, J-H; Liu, Y-B; Wang, J; Liu, L-P; Chen, Y-J; Yang, J; Zhang, Q-H; Wu, Y; Ding, W-J; Hong, T; Xiao, X-H; Zu, X-Y; Wen, G-B

2014-11-27

309

Propiverine-induced accumulation of nuclear and cytosolic protein in F344 rat kidneys: Isolation and identification of the accumulating protein  

SciTech Connect

Male and female F344 rats but not B6C3F1 mice exposed for 104 weeks to propiverine hydrochloride (1-methylpiperid-4-yl 2,2-diphenyl-2-(1-propoxy)acetate hydrochloride), used for treatment of patients with neurogenic detrusor overactivity (NDO) and overactive bladder (OAB), presented with an accumulation of proteins in the cytosol and nuclei of renal proximal tubule epithelial cells, yet despite this, no increased renal tumor incidence was observed. In order to provide an improved interpretation of these findings and a better basis for human health risk assessment, male and female F344 rats were exposed for 16 weeks to 1000 ppm propiverine in the diet, the accumulating protein was isolated from the kidneys via cytosolic and nuclear preparations or laser-capture microdissection and analyzed using molecular weight determination and mass spectrometry. The accumulating protein was found to be D-amino acid oxidase (DAAO), an enzyme involved in amino and fatty acid metabolism. Subsequent reanalysis of kidney homogenate and nuclear samples as well as tissue sections using western blot and DAAO-immunohistochemistry, confirmed the presence and localization of DAAO in propiverine-treated male and female F344 rats. The accumulation of DAAO only in rats, and the limited similarity of rat DAAO with other species, including humans, suggests a rat-specific mechanism underlying the drug-induced renal DAAO accumulation with little relevance for patients chronically treated with propiverine.

Dietrich, D.R. [Environmental Toxicology, University of Konstanz, 78457 Konstanz (Germany)], E-mail: daniel.dietrich@uni-konstanz.de; Heussner, A.H.; O'Brien, E. [Environmental Toxicology, University of Konstanz, 78457 Konstanz (Germany); Gramatte, T.; Runkel, M. [Apogepha Arzneimittel GmbH, 01281 Dresden (Germany); Rumpf, S. [IPMC-TMC GmbH, 8566 Neuwilen (Switzerland); Day, B.W. [Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

2008-12-15

310

Co-localization of the amyloid precursor protein and the Notch intracellular domains in nuclear transcription factories  

PubMed Central

The ?-amyloid precursor protein (APP) plays a major role in Alzheimer’s disease. The APP intracellular domain (AICD), together with Fe65 and Tip60, localizes to spherical nuclear AFT complexes that might represent sites of transcription. We now show that endogenous AICD is targeted to similar nuclear spots. AFT complexes were closely associated with Cajal and PML bodies but did not localize to nucleoli or splicing speckles. Live imaging revealed that AFT complexes were highly mobile within nuclei. Following pharmacological inhibition of transcription AFT complexes merged into a few large assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. Transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at the labeled APP promoter plasmids. In addition, identification of chromosomal APP or KAI1 gene loci by fluorescence in situ hybridization showed their close association with nuclear AFT complexes. The transcriptional activator Notch intracellular domain (NICD) localized to the same nuclear spots as occupied by AFT complexes, suggesting that these nuclear compartments correspond to transcription factories. Fe65 and Tip60 also co-localized with APP in the neurites of primary neurons. Pre-assembled AFT complexes may serve to assist fast nuclear signaling upon endoproteolytic APP cleavage. PMID:18403052

Konietzko, Uwe; Goodger, Zoe V.; Meyer, Michelle; Kohli, Bernhard M.; Bosset, Jerome; Lahiri, Debomoy K.; Nitsch, Roger M.

2009-01-01

311

Synthesis of nuclear proteins during DNA repair synthesis in human diploid fibroblasts damaged with ultraviolet radiation of N-acetoxy-2-acetylaminofluroene.  

PubMed Central

We have examined the accumulation of newly synthesized nuclear proteins into nuclei during DNA repair synthesis in confluent WI-38 human diploid fibroblasts damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluroene. In contrast to a marked stimulation of DNA repair synthesis, stimulation of amino acid incorporation into histone polypeptides or into the various molecular weight classes of nonhistone nuclear proteins was not observed. These results suggest that detectable stimulation of newly synthesized nuclear protein incorporation into nuclei does not accompany DNA repair synthesis induced by ultraviolet radiation or a direct acting chemical carcinogen. At least for the special case of repair, DNA synthesis may be uncoupled from histone synthesis. PMID:1064020

Stein, G S; Park, W D; Stein, J L; Lieberman, M W

1976-01-01

312

Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.  

PubMed

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair. PMID:12754299

Biard, Denis S F; Miccoli, Laurent; Despras, Emmanuelle; Harper, Francis; Pichard, Evelyne; Créminon, Christophe; Angulo, Jaime F

2003-05-01

313

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells  

Microsoft Academic Search

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor ? (ER?) and\\u000a estrogen receptor ? (ER?) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen\\u000a receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation.\\u000a The aim of our

Roman Pavlik; Gabriela Wypior; Stefanie Hecht; Panos Papadopoulos; Markus Kupka; Christian Thaler; Irmi Wiest; Aurelia Pestka; Klaus Friese; Udo Jeschke

314

A study of the nuclear trafficking of the splicing factor protein PRPF31 linked to autosomal dominant retinitis pigmentosa (ADRP)  

Microsoft Academic Search

In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin ?1 for translocation to the nucleus, with no requirement for importin ?1. The A194E and A216P mutations have no

Susan E. Wilkie; Keith J. Morris; Shomi S. Bhattacharya; Martin J. Warren; David M. Hunt

2006-01-01

315

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex  

Microsoft Academic Search

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex

J. O. Bustamante; A. Liepins; R. A. Prendergast; J. A. Hanover; H. Oberleithner

1995-01-01

316

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system  

Microsoft Academic Search

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori

Tomoko Motohashi; Tsukasa Shimojima; Tatsuo Fukagawa; Katsumi Maenaka; Enoch Y. Park

2005-01-01

317

Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes  

PubMed Central

Barrier to autointegration factor (BAF), which is encoded by the BANF1 gene, binds with high-affinity to double-stranded DNA and LEM domain–containing proteins at the nuclear periphery. A BANF1 mutation has recently been associated with a novel human progeria syndrome, and cells from these patients have aberrant nuclear envelopes. The interactions of BAF with its DNA- and protein-binding partners are known to be regulated by phosphorylation, and previously we validated BAF as a highly efficient substrate for the VRK1 protein kinase. Here we show that depletion of VRK1 in MCF10a and MDA-MB-231 cells results in aberrant nuclear architecture. The immobile fraction of green fluorescent protein (GFP)–BAF at the nuclear envelope (NE) is elevated, suggesting that prolonged interactions of BAF with its binding partners is likely responsible for the aberrant NE architecture. Because detachment of BAF from its binding partners is associated with NE disassembly, we performed live-imaging analysis of control and VRK1-depleted cells to visualize GFP-BAF dynamics during mitosis. In the absence of VRK1, BAF does not disperse but instead remains chromosome bound from the onset of mitosis. VRK1 depletion also increases the number of anaphase bridges and multipolar spindles. Thus phosphorylation of BAF by VRK1 is essential both for normal NE architecture and proper dynamics of BAF–chromosome interactions during mitosis. These results are consistent with previous studies of the VRK/BAF signaling axis in Caenorhabditis elegans and Drosophila melanogaster and validate VRK1 as a key regulator of NE architecture and mitotic chromosome dynamics in mammalian cells. PMID:24430874

Molitor, Tyler P.; Traktman, Paula

2014-01-01

318

Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes.  

PubMed

Barrier to autointegration factor (BAF), which is encoded by the BANF1 gene, binds with high-affinity to double-stranded DNA and LEM domain-containing proteins at the nuclear periphery. A BANF1 mutation has recently been associated with a novel human progeria syndrome, and cells from these patients have aberrant nuclear envelopes. The interactions of BAF with its DNA- and protein-binding partners are known to be regulated by phosphorylation, and previously we validated BAF as a highly efficient substrate for the VRK1 protein kinase. Here we show that depletion of VRK1 in MCF10a and MDA-MB-231 cells results in aberrant nuclear architecture. The immobile fraction of green fluorescent protein (GFP)-BAF at the nuclear envelope (NE) is elevated, suggesting that prolonged interactions of BAF with its binding partners is likely responsible for the aberrant NE architecture. Because detachment of BAF from its binding partners is associated with NE disassembly, we performed live-imaging analysis of control and VRK1-depleted cells to visualize GFP-BAF dynamics during mitosis. In the absence of VRK1, BAF does not disperse but instead remains chromosome bound from the onset of mitosis. VRK1 depletion also increases the number of anaphase bridges and multipolar spindles. Thus phosphorylation of BAF by VRK1 is essential both for normal NE architecture and proper dynamics of BAF-chromosome interactions during mitosis. These results are consistent with previous studies of the VRK/BAF signaling axis in Caenorhabditis elegans and Drosophila melanogaster and validate VRK1 as a key regulator of NE architecture and mitotic chromosome dynamics in mammalian cells. PMID:24430874

Molitor, Tyler P; Traktman, Paula

2014-03-01

319

The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes  

Microsoft Academic Search

Squamate reptiles number approximately 8000 living species and are a major component of the world's terrestrial vertebrate diversity. However, the established relationships of the higher-level groups have been questioned in recent molecular analyses. Here we expand the molecular data to include DNA sequences, totaling 6192 base pairs (bp), from nine nuclear protein-coding genes (C-mos, RAG1, RAG2, R35, HOXA13, JUN, ?-enolase,

Nicolas Vidal; S. Blair Hedges

2005-01-01

320

[Interaction of the new gestagen compound 17alpha-acetoxy-3beta-butanoyloxy-6-methylpregna-4,6-dien-20-one with human blood proteins and calf embryo serum].  

PubMed

Molecular mechanisms of sex hormones (progesterone, medroxyprogesterone acetate (MPA), and new synthetic gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methylpregna-4,6-dien-20-on (ABMP) with human serum albumin, globulins, and calf embryo serum were studied. The binding of ABMP to albumin and progesterone-binding proteins were investigated using spectroscopic techniques (with the use of 1-anilino-8-naphthalenesulfonate as fluorescent probe) and radiolabeled progesterone, (either progesteron or MPA as comparative progestines). There is no difference between the non-specific binding of ABMP, progesterone, and MPA, but the ABMP binding is much smaller as compared to the binding of progestines, so that the new progestine ABMP will produce a more effective action on the target tissue than comparative progestines. PMID:18488905

Vetchinkina, V B; Seme?kin, A V; Rzheznikov, V M; Shimanovski?, N L

2008-01-01

321

The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration  

PubMed Central

Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect—live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus. PMID:25057012

Bone, Courtney R.; Tapley, Erin C.; Gorjánácz, Mátyás; Starr, Daniel A.

2014-01-01

322

The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration.  

PubMed

Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect-live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus. PMID:25057012

Bone, Courtney R; Tapley, Erin C; Gorjánácz, Mátyás; Starr, Daniel A

2014-09-15

323

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.  

PubMed Central

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC. Images PMID:8443417

Loeb, J D; Davis, L I; Fink, G R

1993-01-01

324

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans  

SciTech Connect

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.

Ji Qiongmei [Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, NY 10021 (United States); Huang, C.-H. [Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, NY 10021 (United States)]. E-mail: chuang@nybloodcenter.org; Peng Jianbin [Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, NY 10021 (United States); Hashmi, Sarwar [Developmental Biology, Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States); Ye Tianzhang [Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, NY 10021 (United States); Chen Ying [Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, NY 10021 (United States)

2007-04-15

325

Nuclear  

NSDL National Science Digital Library

What part does nuclear energy play in satisfying energy demands? This informational piece, part of a series about the future of energy, introduces students to the uranium atom as an energy source. Here students read about the history of nuclear energy, how energy is derived from uranium, and benefits of nuclear energy. Information is also provided about limitations, particularly disposal problems and radioactivity, and geographical considerations of nuclear power in the United States. Thought-provoking questions afford students chances to reflect on what they've read about the uses of nuclear power. Articles and information on new nuclear plant design and nuclear accidents are available from a sidebar. Five energy-related PBS NewsHour links are provided. A web link to the U.S. Nuclear Regulatory Commission is included. Copyright 2005 Eisenhower National Clearinghouse

Project, Iowa P.

2004-01-01

326

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments  

PubMed Central

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns. PMID:24391881

Nisa-Martinez, Rafael; Laporte, Philippe; Jimenez-Zurdo, Jose Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolas

2013-01-01

327

Purifying selection in mammalian mitochondrial protein-coding genes is highly effective and congruent with evolution of nuclear genes.  

PubMed

The mammalian mitochondrial genomes differ from the nuclear genomes by maternal inheritance, absence of recombination, and higher mutation rate. All these differences decrease the effective population size of mitochondrial genome and make it more susceptible to accumulation of slightly deleterious mutations. It was hypothesized that mitochondrial genes, especially in species with low effective population size, irreversibly degrade leading to decrease of organismal fitness and even to extinction of species through the mutational meltdown. To interrogate this hypothesis, we compared the purifying selections acting on the representative set of mitochondrial (potentially degrading) and nuclear (potentially not degrading) protein-coding genes in species with different effective population size. For 21 mammalian species, we calculated the ratios of accumulation of slightly deleterious mutations approximated by Kn/Ks separately for mitochondrial and nuclear genomes. The 75% of variation in Kn/Ks is explained by two independent variables: type of a genome (mitochondrial or nuclear) and effective population size of species approximated by generation time. First, we observed that purifying selection is more effective in mitochondria than in the nucleus that implies strong evolutionary constraints of mitochondrial genome. Mitochondrial de novo nonsynonymous mutations have at least 5-fold more harmful effect when compared with nuclear. Second, Kn/Ks of mitochondrial and nuclear genomes is positively correlated with generation time of species, indicating relaxation of purifying selection with decrease of species-specific effective population size. Most importantly, the linear regression lines of mitochondrial and nuclear Kn/Ks's from generation times of species are parallel, indicating congruent relaxation of purifying selection in both genomes. Thus, our results reveal that the distribution of selection coefficients of de novo nonsynonymous mitochondrial mutations has a similar shape with the distribution of de novo nonsynonymous nuclear mutations, but its mean is five times smaller. The harmful effect of mitochondrial de novo nonsynonymous mutations triggers highly effective purifying selection, which maintains the fitness of the mammalian mitochondrial genome. PMID:22983951

Popadin, Konstantin Yu; Nikolaev, Sergey I; Junier, Thomas; Baranova, Maria; Antonarakis, Stylianos E

2013-02-01

328

Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences.  

PubMed

The remarkable antiquity, diversity and ecological significance of arthropods have inspired numerous attempts to resolve their deep phylogenetic history, but the results of two decades of intensive molecular phylogenetics have been mixed. The discovery that terrestrial insects (Hexapoda) are more closely related to aquatic Crustacea than to the terrestrial centipedes and millipedes (Myriapoda) was an early, if exceptional, success. More typically, analyses based on limited samples of taxa and genes have generated results that are inconsistent, weakly supported and highly sensitive to analytical conditions. Here we present strongly supported results from likelihood, Bayesian and parsimony analyses of over 41 kilobases of aligned DNA sequence from 62 single-copy nuclear protein-coding genes from 75 arthropod species. These species represent every major arthropod lineage, plus five species of tardigrades and onychophorans as outgroups. Our results strongly support Pancrustacea (Hexapoda plus Crustacea) but also strongly favour the traditional morphology-based Mandibulata (Myriapoda plus Pancrustacea) over the molecule-based Paradoxopoda (Myriapoda plus Chelicerata). In addition to Hexapoda, Pancrustacea includes three major extant lineages of 'crustaceans', each spanning a significant range of morphological disparity. These are Oligostraca (ostracods, mystacocarids, branchiurans and pentastomids), Vericrustacea (malacostracans, thecostracans, copepods and branchiopods) and Xenocarida (cephalocarids and remipedes). Finally, within Pancrustacea we identify Xenocarida as the long-sought sister group to the Hexapoda, a result confirming that 'crustaceans' are not monophyletic. These results provide a statistically well-supported phylogenetic framework for the largest animal phylum and represent a step towards ending the often-heated, century-long debate on arthropod relationships. PMID:20147900

Regier, Jerome C; Shultz, Jeffrey W; Zwick, Andreas; Hussey, April; Ball, Bernard; Wetzer, Regina; Martin, Joel W; Cunningham, Clifford W

2010-02-25

329

Transcriptional regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear factor-4alpha.  

PubMed

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly of triglyceride (TG)-rich apolipoprotein B-containing liver (e.g., VLDL) and intestinal (e.g., chylomicron) lipoproteins. The human MTP gene promoter is reported here to associate in vivo with endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) and to be transactivated or transsuppressed by overexpressed or by dominant negative HNF-4alpha, respectively. Human MTP (hMTP) transactivation by HNF-4alpha is accounted for by the concerted activity of distal (-83/-70) and proximal (-50/-38) direct repeat 1 elements of the hMTP promoter that bind HNF-4alpha. Transactivation by HNF-4alpha is specifically antagonized by chicken ovalbumin upstream promoter. Transcriptional activation of hMTP by HNF-4alpha is mediated by HNF-4alpha domains engaged in ligand binding and ligand-driven transactivation and is further complemented by HNF-4alpha/HNF-1alpha synergism that involves the HNF-4alpha activation function 1 (AF-1) domain. hMTP transactivation by HNF-4alpha is specifically inhibited by beta,beta-tetramethyl-hexadecanedioic acid acting as an HNF-4alpha antagonist ligand. hMTP transactivation by HNF-4alpha may account for the activation or inhibition of MTP expression and the production of TG-rich lipoproteins by agonist (e.g., saturated fatty acids) or antagonist [e.g., (n-3) PUFA, hypolipidemic fibrates, or Methyl-substituted dicarboxylic acid (Medica) compounds] HNF-4alpha ligands. PMID:15547294

Sheena, Vered; Hertz, Rachel; Nousbeck, Janna; Berman, Ina; Magenheim, Judith; Bar-Tana, Jacob

2005-02-01

330

Nuclear proteins from Drosophila melanogaster embryos which specifically bind to simple homopolymeric sequences poly [(dT-dG).(dC-dA)].  

PubMed Central

The binding of nuclear proteins from Drosophila melanogaster embryos to simple homopolymeric DNA sequences was studied. Nuclear proteins were electrophoresed, transferred onto nitrocellulose and incubated with labelled synthetic homopolymers or natural fragment containing simple sequences. Several protein bands were found in the 65-72 KDa region, which specifically bind both poly [(dG-dT).(dA-dC)] and a natural fragment containing 40 bp of this sequence. These proteins do not bind to homopolymers poly [(dA).(dT)] and poly [(dG-dA).(dC-dT)], or other foreign DNAs. Images PMID:3133638

Vashakidze, R P; Chelidze, M G; Mamulashvili, N A; Kalandarishvili, K G; Tsalkalamanidze, N V

1988-01-01

331

Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number.  

PubMed Central

The mitochondrial Rieske iron-sulfur protein is an obligatory component of the respiratory electron transport chain that is encoded by a single-copy gene in mammals and fungi. In contrast, this protein is encoded by a small gene family in dicotyledonous tobacco and monocotyledonous maize. We cloned four cDNAs from tobacco that encode the mitochondrial Rieske iron-sulfur protein. These clones, along with a previously isolated cDNA, represent five independent members of the gene family that can be divided into three subfamilies. All of these genes were derived from the two progenitor species and were expressed in amphidiploid tobacco. The proteins encoded by these five genes are probably functional because they all contain the universally conserved hexyl peptides necessary for the 2Fe-2S cluster formation. The expression of the Rieske protein gene family is differentially regulated; a 6- to 11-fold higher level of steady state transcripts was found in flowers than in leaves, stems, and roots. Members of at least two subfamilies were preferentially expressed in flowers, indicating that they share a common cis-regulatory element(s), which can respond to a flower-specific signal(s). Although approximately 10 times more transcripts occurred in flowers than in leaves, flower and leaf mitochondria contained a similar amount of the Rieske protein. Flowers, however, contained seven times more Rieske proteins than leaves. These results indicated an increase in mitochondrion number in flowers. High-energy demands during anther development might bring about an increase in mitochondrion numbers in flowers and the flower-enhanced expression of the Rieske protein gene family. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism and/or changes in mitochondrion numbers. PMID:8180500

Huang, J; Struck, F; Matzinger, D F; Levings, C S

1994-01-01

332

Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13  

SciTech Connect

A monoclonal antibody that was specific for a nuclear matrix protein was obtained and used to screen a human [lambda]gt11 expression library. Several partial cDNA clones were isolated and sequenced. The sequence for this protein was shown to be identical to that of NuMA, a 236-kDa nuclear mitotic spindle apparatus protein. NuMA has been recently characterized by two independent studies, and is thought to be part of a family of proteins that is required for the completion of mitosis. In this report, the chromosomal localization and copy number of the NuMA gene are analyzed using cDNA clones. High-resolution in situ hybridization reveals a single pair of signals on sister chromatids of human chromosome 11 at band q13. Stringent Southern analysis of human genomic DNA resulted in simple restriction patterns. These results together indicate that the NuMA gene is present as a single copy on human chromosome 11q13. 12 refs., 1 fig.

Sparks, C.A.; Bangs, P.L.; Lawrence, J.B.; Fey, E.G.; McNeil, G.P. (Univ. of Massachusetts Medical School, Worcester, MA (United States))

1993-07-01

333

A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo  

PubMed Central

A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport. PMID:2459127

1988-01-01

334

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation  

PubMed Central

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF. PMID:19648179

Kim, Kyoung Mi; Cho, Hana; Choi, Kobong; Kim, Jaedong; Kim, Bong-Woo; Ko, Young-Gyu; Jang, Sung Key; Kim, Yoon Ki

2009-01-01

335

Expression of nuclear lamin A and muscle-specific proteins in differentiating muscle cells in ovo and in vitro  

PubMed Central

Primary cultures and tissue samples of chicken embryonic muscle were immunologically probed for the expression of muscle-specific proteins, such as myosin heavy chain and the tropomyosins, as well as for the nuclear lamina protein, lamin A. As determined by quantitative immunoblotting, the expression of lamin A and the muscle-specific proteins were at low levels or absent in predifferentiation myoblasts both in vitro and in ovo. During differentiation, an increase of lamin A expression preceded the induction to high levels of expression of muscle-specific proteins. Immunofluorescence staining of chicken embryonic muscle cells in culture also indicates an accumulation of lamin A before the induction of muscle-specific proteins expression. Furthermore, the accumulation of lamin A reached a plateau before the muscle-specific proteins during muscle development. In two dimensional NEPHGE gel analysis of immunoprecipitated lamin A, no detectable change in the ratio of the acidic/basic isoelectric variants of lamin A was observed during myogenesis. A potential role for lamin A in the mechanisms which underlie the differential and coordinate expression of muscle-specific genes is proposed. PMID:2668298

1989-01-01

336

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.  

PubMed

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals. PMID:15289615

Kim, Sung Bae; Ozawa, Takeaki; Watanabe, Shigeaki; Umezawa, Yoshio

2004-08-10

337

Activation of nuclear transcription factor-kappa B is associated with the induction of inhibitory kappa B kinase-beta and involves differential activation of protein kinase C and protein tyrosine kinases during fatal murine cerebral malaria  

Microsoft Academic Search

The levels of nuclear transcription factor-kappa B (NF-?B) subunits p65 and p50 and its associated kinase, inhibitory kappa B kinase (IKK) alpha and beta were monitored in cytosolic and nuclear fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Since protein kinase C (PKC) and protein tyrosine kinases (PTK) are known to

Kota Arun Kumar; Yadavalli Rajgopal; Usha Pillai; Phanithi Prakash Babu

2003-01-01

338

Electrophoretic characterization of nuclear basic proteins in the toxic marine dinoflagellate Gymnodinium mikimotoi  

E-print Network

protein spots, consistent with those seen previously in Crypthecodinium cohnii. HGm variants responded to nitrogen stress and time of day, but did not respond to other environmental variations. Therefore, while the protein was further characterized, its...

Wargo, Matthew James

2012-06-07

339

The quantitative assessment of the role played by basic amino acid clusters in the nuclear uptake of human ribosomal protein L7  

SciTech Connect

In this study, we used a multiple copy (EGFP){sub 3} reporter system to establish a numeric nuclear index system to assess the degree of nuclear import. The system was first validated by a FRAP assay, and then was applied to evaluate the essential and multifaceted nature of basic amino acid clusters during the nuclear import of ribosomal protein L7. The results indicate that the sequence context of the basic cluster determines the degree of nuclear import, and that the number of basic residues in the cluster is irrelevant; rather the position of the pertinent basic residues is crucial. Moreover, it also found that the type of carrier protein used by basic cluster has a great impact on the degree of nuclear import. In case of L7, importin ?2 or importin ?3 are preferentially used by clusters with a high import efficiency, notwithstanding that other importins are also used by clusters with a weaker level of nuclear import. Such a preferential usage of multiple basic clusters and importins to gain nuclear entry would seem to be a common practice among ribosomal proteins in order to ensure their full participation in high rate ribosome synthesis. - Highlights: ? We introduce a numeric index system that represents the degree of nuclear import. ? The rate of nuclear import is dictated by the sequence context of the basic cluster. ? Importin ?2 and ?3 were mainly responsible for the N4 mediated nuclear import.

Tai, Lin-Ru [Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Chou, Chang-Wei [Institute of Clinical Dentistry Science, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lee, I-Fang; Kirby, Ralph [Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lin, Alan, E-mail: alin@ym.edu.tw [Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Institute of Clinical Dentistry Science, National Yang-Ming University, Taipei, Taiwan, ROC (China)

2013-02-15

340

Deciphering apicoplast targeting signals feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins  

E-print Network

of Plasmodium falciparum apicoplast proteins Jochen Zueggea,b , Stuart Ralphc , Michael Schmukera , Geoffrey I Plasmodium falciparum contains a vestigal, non-photosynthetic plastid, the apicoplast. Numerous proteins tools in place for prediction of subcellular locations for all proteins. Apicoplast targeting signals

McFadden, Geoff

341

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization  

Microsoft Academic Search

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil-forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have

Manuela E. Hase; Nikolai V. Kuznetsov; Volker C. Cordes

2001-01-01

342

Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2  

PubMed Central

Background Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA) expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. Methodology/Principal Findings Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS) to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360). Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C2C12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C2C12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. Conclusions/Significance Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status likely control nucleocytoplasmic localization of IRF2BP2 during muscle differentiation. PMID:21887377

Teng, Allen C. T.; Al-montashiri, Naif A. M.; Cheng, Brian L. M.; Lou, Philip; Ozmizrak, Pinar; Chen, Hsiao-Huei; Stewart, Alexandre F. R.

2011-01-01

343

Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes*  

PubMed Central

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics. PMID:19933576

Haque, Farhana; Mazzeo, Daniela; Patel, Jennifer T.; Smallwood, Dawn T.; Ellis, Juliet A.; Shanahan, Catherine M.; Shackleton, Sue

2010-01-01

344

Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite.  

PubMed

The inhalation of asbestos is a risk factor for the development of malignant mesothelioma and lung cancer. Based on the broad surface area of asbestos fibers and their ability to enter the cytoplasm and nuclei of cells, it was hypothesized that proteins that adsorb onto the fiber surface play a role in the cytotoxicity and carcinogenesis of asbestos fibers. However, little is known about which proteins adsorb onto asbestos. Previously, we systematically identified asbestos-interacting proteins and classified them into eight sub-categories: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. Here, we report an adsorption profile of proteins for the three commercially used asbestos compounds: chrysotile, crocidolite and amosite. We quantified the amounts of adsorbed proteins by analyzing the silver-stained gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis with ImageJ software, using the bands for amosite as a standard. We found that histones were most adsorptive to crocidolite and that chromatin-binding proteins were most adsorptive to chrysotile. The results suggest that chrysotile and crocidolite directly interact with chromatin structure through different mechanisms. Furthermore, RNA-binding proteins preferably interacted with chrysotile, suggesting that chrysotile may interfere with transcription and translation. Our results provide novel evidence demonstrating that the specific molecular interactions leading to carcinogenesis are different between chrysotile and crocidolite. PMID:23170051

Kubo, Yurika; Takenaka, Hiroyuki; Nagai, Hirotaka; Toyokuni, Shinya

2012-11-01

345

Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.  

PubMed

Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3? protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3? ligand binding site are responsible for the binding of 14-3-3? to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3?. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3? following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A. PMID:23395486

Conti, Audrey; Sueur, Charlotte; Lupo, Julien; Brazzolotto, Xavier; Burmeister, Wim P; Manet, Evelyne; Gruffat, Henri; Morand, Patrice; Boyer, Véronique

2013-03-01

346

Nuclear Eg5 (kinesin spindle protein) expression predicts docetaxel response and prostate cancer aggressiveness  

PubMed Central

Novel biomarkers predicting prostate cancer (PCa) aggressiveness and docetaxel therapy response of PCa patients are needed. In this study the correlation between nuclear Eg5-expression, PCa docetaxel response and PCa aggressiveness was assessed. Immunohistochemical staining for nuclear Eg5 was performed on 117 archival specimens from 110 PCa patients treated with docetaxel between 2004 and 2012. Samples were histologically categorized as positive/negative. Median follow-up time from diagnosis was 11.6 years. Nuclear Eg5-expression was significantly related to docetaxel response (p=0.036) in tissues acquired within three years before docetaxel initiation. Nuclear Eg5-expression was not related to Gleason-score (p=0.994). Survival of patients after docetaxel initiation did not differ based on nuclear Eg5-expression (p=0.540). Analyzing samples taken before hormonal therapy, overall survival and time to docetaxel use were significantly decreased in patients with nuclear Eg5-expressing tumors (p<0.01). Eg5-positive nuclei were found more frequently in T4-staged tumors (p=0.04), Gleason 8-10 tumors (p=0.08), and in metastasized tumors (p<0.01). Multivariate analyses indicated that nuclear Eg5-expression may be an independent parameter for tumor aggressiveness. Limitations of a retrospective analysis apply. In conclusion, nuclear Eg5-expression may be a predictive biomarker for docetaxel response in metastatic castrate-resistant PCa patients and a prognostic biomarker for hormone-naive PCa patients. Prospective validation studies are needed. PMID:25277178

Dezentje, Vincent O.; Buijs, Jeroen T.; De Krijger, Ronald R.; Smit, Vincent T.H.B.M.; Van Weerden, Wytske M.; Gelderblom, Hans; van der Pluijm, Gabri

2014-01-01

347

Heat Shock Protein 83 (Hsp83) Facilitates Methoprene-tolerant (Met) Nuclear Import to Modulate Juvenile Hormone Signaling.  

PubMed

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1? promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR. PMID:25122763

He, Qianyu; Wen, Di; Jia, Qiangqiang; Cui, Chunlai; Wang, Jian; Palli, Subba R; Li, Sheng

2014-10-01

348

Regulation of Hepatitis C Virus Replication by Nuclear Translocation of Nonstructural 5A Protein and Transcriptional Activation of Host Genes  

PubMed Central

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is involved in regulating viral replication through its direct interaction with the HCV RNA-dependent RNA polymerase. NS5A also alters infected cell metabolism through complex interactions with numerous host cell proteins. NS5A has furthermore been suggested to act as a transcriptional activator, although the impact on viral replication is unclear. To study this, HCV NS5A variants were amplified from hepatic tissue from an HCV-infected patient, and their abilities to activate gene transcription were analyzed in a single-hybrid yeast (Saccharomyces cerevisiae) model. Different variants isolated from the same patient displayed different transactivational activities. When these variants were inserted into the HCV subgenomic replicon system, they demonstrated various levels of RNA replication, which correlated with their transactivational activities. We showed that the C-terminal fragment of NS5A was localized to the nucleus and that a functional NS5A nuclear localization signal and cellular caspase activity were required for this process. Furthermore, nuclear localization of NS5A was necessary for viral replication. Finally, we demonstrate that nuclear NS5A binds to host cell promoters of several genes previously identified as important for efficient HCV RNA replication, inducing their transcription. Taken together, these results demonstrate a new mechanism by which HCV modulates its cellular environment, thereby enhancing viral replication. PMID:23468497

Maqbool, Muhammad Ahmad; Imache, Mohamed R.; Higgs, Martin R.; Carmouse, Sophie; Pawlotsky, Jean-Michel

2013-01-01

349

Mitochondrial reactive oxygen species are scavenged by Cockayne syndrome B protein in human fibroblasts without nuclear DNA damage.  

PubMed

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role. PMID:25136123

Cleaver, James E; Brennan-Minnella, Angela M; Swanson, Raymond A; Fong, Ka-Wing; Chen, Junjie; Chou, Kai-Ming; Chen, Yih-Wen; Revet, Ingrid; Bezrookove, Vladimir

2014-09-16

350

Saccharomyces cerevisiae MPS2 Encodes a Membrane Protein Localized at the Spindle Pole Body and the Nuclear Envelope  

PubMed Central

The MPS2 (monopolar spindle two) gene is one of several genes required for the proper execution of spindle pole body (SPB) duplication in the budding yeast Saccharomyces cerevisiae (Winey et al., 1991). We report here that the MPS2 gene encodes an essential 44-kDa protein with two putative coiled-coil regions and a hydrophobic sequence. Although MPS2 is required for normal mitotic growth, some null strains can survive; these survivors exhibit slow growth and abnormal ploidy. The MPS2 protein was tagged with nine copies of the myc epitope, and biochemical fractionation experiments show that it is an integral membrane protein. Visualization of a green fluorescent protein (GFP) Mps2p fusion protein in living cells and indirect immunofluorescence microscopy of 9xmyc-Mps2p revealed a perinuclear localization with one or two brighter foci of staining corresponding to the SPB. Additionally, immunoelectron microscopy shows that GFP-Mps2p localizes to the SPB. Our analysis suggests that Mps2p is required as a component of the SPB for insertion of the nascent SPB into the nuclear envelope. PMID:10397772

Munoz-Centeno, Maria de la Cruz; McBratney, Susan; Monterrosa, Antonio; Byers, Breck; Mann, Carl; Winey, Mark

1999-01-01

351

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.  

PubMed

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein. PMID:169239

Oldfield, E; Norton, R S; Allerhand, A

1975-08-25

352

p35 interacts with alpha-tubulin and organelle proteins: nuclear translocation of p35 in dying cells.  

PubMed

We identified heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, hnRNP A1, the translocase of the transporter outer membrane 40 (TOM40), and alpha-tubulin as new interaction partners of anti-apoptotic protein p35 using MS-based functional proteomics with GST-p35 fusion protein as a bait, and using a pull-down assay with p35-6His followed by Western blot analysis. p35 was localized in the cytoplasm and in distinct organelles such as the nucleus and mitochondria. p35 was more abundant in the cytoplasm than it was in the nucleus. It co-localized with alpha-tubulin in the cytoplasm in the absence of a death stimulus. However, while cells were undergoing death induced by actinomycin D, cytoplasmic p35 was translocated into the nucleus; this process was inhibited by deletions of the N- and C-terminal domains containing leucine-rich motifs. Gene delivery of p35 using recombinant adenoviruses inhibited cytoplasmic compartmentalization of hnRNP C1/C2 and hnRNP A1 in dying cells. This study demonstrated translocation of p35 into the nuclei, as well as protection of the hnRNPs from redistribution in cells undergoing death. We propose an active role for p35 in maintaining the integrity of nuclear proteins during cell death. PMID:19701913

Son, Yonghae; Kim, Sunmi; Choi, Kyungha; Park, Youngchul; Eo, Seongkug; Kim, Youngkook; Rhim, Byungyong; Kim, Koanhoi

2009-08-01

353

Interaction of nucleosome assembly proteins abolishes nuclear localization of DGK{zeta} by attenuating its association with importins  

SciTech Connect

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGK{zeta}, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGK{zeta}. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGK{zeta} binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGK{zeta} and NAP1Ls prohibits nuclear import of DGK{zeta} because binding of NAP1Ls to DGK{zeta} blocks import carrier proteins, Qip1 and NPI1, to interact with DGK{zeta}, leading to cytoplasmic tethering of DGK{zeta}. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK{zeta} and provide a clue to examine functional significance of its translocation under pathological conditions.

Okada, Masashi; Hozumi, Yasukazu [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)] [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Ichimura, Tohru [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan)] [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan); Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)] [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Iseki, Ken [Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)] [Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Yagisawa, Hitoshi [Laboratory of Biological Signaling, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297 (Japan)] [Laboratory of Biological Signaling, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297 (Japan); Shinkawa, Takashi; Isobe, Toshiaki [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan)] [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan); Goto, Kaoru, E-mail: kgoto@med.id.yamagata-u.ac.jp [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)] [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)

2011-12-10

354

Global human frequencies of predicted nuclear pathogenic variants and the role played by protein hydrophobicity in pathogenicity potential.  

PubMed

Mitochondrial proteins are coded by nuclear (nDNA) and mitochondrial (mtDNA) genes, implying a complex cross-talk between the two genomes. Here we investigated the diversity displayed in 104 nuclear-coded mitochondrial proteins from 1,092 individuals from the 1000 Genomes dataset, in order to evaluate if these genes are under the effects of purifying selection and how that selection compares with their mitochondrial encoded counterparts. Only the very rare variants (frequency < 0.1%) in these nDNA genes are indistinguishable from a random set from all possible variants in terms of predicted pathogenicity score, but more frequent variants display distinct signs of purifying selection. Comparisons of selection strength indicate stronger selection in the mtDNA genes compared to this set of nDNA genes, accounted for by the high hydrophobicity of the proteins coded by the mtDNA. Most of the predicted pathogenic variants in the nDNA genes were restricted to a single continental population. The proportion of individuals having at least one potential pathogenic mutation in this gene set was significantly lower in Europeans than in Africans and Asians. This difference may reflect demographic asymmetries, since African and Asian populations experienced main expansions in middle Holocene, while in Europeans the main expansions occurred earlier in the post-glacial period. PMID:25412673

Pereira, Luísa; Soares, Pedro; Triska, Petr; Rito, Teresa; van der Waerden, Agnes; Li, Biao; Radivojac, Predrag; Samuels, David C

2014-01-01

355

Mitogenic and Oncogenic Stimulation of K433 Acetylation Promotes PKM2 Protein Kinase Activity and Nuclear Localization  

PubMed Central

SUMMARY Alternative splicing of the PKM2 gene produces two isoforms, M1 and M2, which are preferentially expressed in adult and embryonic tissues, respectively. The M2 isoform is reexpressed in human cancer and has nonmetabolic functions in the nucleus as a protein kinase. Here, we report that PKM2 is acetylated by p300 acetyltransferase at K433, which is unique to PKM2 and directly contacts its allosteric activator, fructose 1,6-bisphosphate (FBP). Acetylation prevents PKM2 activation by interfering with FBP binding and promotes the nuclear accumulation and protein kinase activity of PKM2. Acetylationmimetic PKM2(K433) mutant promotes cell proliferation and tumorigenesis. K433 acetylation is decreased by serum starvation and cell-cell contact, increased by cell cycle stimulation, epidermal growth factor (EGF), and oncoprotein E7, and enriched in breast cancers. Hence, K433 acetylation links cell proliferation and transformation to the switch of PKM2 from a cytoplasmic metabolite kinase to a nuclear protein kinase. PMID:24120661

Lv, Lei; Xu, Yan-Ping; Zhao, Di; Li, Fu-Long; Wang, Wei; Sasaki, Naoya; Jiang, Ying; Zhou, Xin; Li, Ting-Ting; Guan, Kun-Liang; Lei, Qun-Ying; Xiong, Yue

2014-01-01

356

An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway  

PubMed Central

In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei —except the fibrillar center of nucleolus— and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes. PMID:21687672

Serpeloni, Mariana; Moraes, Carolina Borsoi; Muniz, Joao Renato Carvalho; Motta, Maria Cristina Machado; Ramos, Augusto Savio Peixoto; Kessler, Rafael Luis; Inoue, Alexandre Haruo; Duarte daRocha, Wanderson; Yamada-Ogatta, Sueli Fumie; Fragoso, Stenio Perdigao; Goldenberg, Samuel; Freitas-Junior, Lucio H.; Avila, Andrea Rodrigues

2011-01-01

357

Expression of RNUDC, a Potential Nuclear Movement Protein, in Mammalian Cells: Localization to the Golgi Apparatus  

Microsoft Academic Search

Prolactin and other cytokines regulate lymphocyte proliferation through the activation of a number of genes, one of which was identified asRnudCfrom a prolactin-dependent rat T cell line, Nb2.RnudCencodes a 45-kDa protein whose carboxy terminal 94 amino acids are similar to the carboxy terminus of theAspergillus nidulansnuclear movement protein NUDC. In Nb2 T cells, RNUDC protein levels are induced two- to

Shelli M. Morris; Li-yuan Yu-Lee

1998-01-01

358

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins  

Microsoft Academic Search

Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector ?gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting

Jochen Tittgen; Jiirgen Hermans; Johannes Steppuhn; Thomas Jansen; Christer Jansson; Bertil Andersson; Rachel Nechushtai; Nathan Nelson; Reinhold G. Herrmann

1986-01-01

359

Proteins.  

ERIC Educational Resources Information Center

Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

Doolittle, Russell F.

1985-01-01

360

Proteins  

NSDL National Science Digital Library

Laboratory manual and supplemental resources that were developed for a college laboratory course in protein purification. The enzyme, Beta-galactosidase, is purified in two steps, with analysis and verification of results. Course materials are divided into four units: Why Proteins, Assays, The Purification Process, and Analysis and Verification. Powerpoint lectures and study guides are provided.

Mowery, Jeanette; Seidman, Lisa A.

2009-10-01