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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

Nuclear Protein Transport Pathways  

Microsoft Academic Search

Nuclear proteins like transcription factors and ribosomal proteins are synthesized in the cytoplasm and have to be transported into the nucleus to fulfill their functions. The transport of proteins >20–60 kD through the nuclear pore complex (NPC) into the nucleus is an active, energy-requiring process. Transport substrates are recognized by their transport proteins via certain signals. The best-characterized protein import

Matthias Köhler; Hermann Haller; Enno Hartmann

1999-01-01

3

Progesterone Binding to the ?1-Subunit of the Na/K-ATPase on the Cell Surface: Insights from Computational Modeling  

PubMed Central

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [3H]ouabain and [3H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112 kDa ?1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding ?1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar ? and hydrophobic ? surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg118, Asp129 and possibly Glu122-124 in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys111 near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding. PMID:17936318

Morrill, Gene A.; Kostellow, Adele B.; Askari, Amir

2008-01-01

4

HMGA proteins: flexibility finds a nuclear niche?  

PubMed

The mammalian HMGA family of chromatin proteins possesses an unusual constellation of physical, biochemical, and biological characteristics that distinguish them from other nuclear proteins. Principal among these is the fact that, unlike other proteins, they possess little detectable secondary structure prior to interactions with other macromolecules (DNA, RNA, proteins). Upon binding to substrates, however, the HMGA proteins undergo specific disordered-to-ordered structural transitions and also induce alterations in the structure of the substrates themselves. Their intrinsic structural flexibility, combined with other features such as the control of their substrate interactions via complex patterns of in vivo biochemical modifications, allows the HMGA proteins to actively participate in a wide variety of nuclear activities including DNA replication, DNA repair, chromatin remodeling, control of gene transcription, and regulation of mRNA processing PMID:12897853

Reeves, Raymond

2003-06-01

5

Protein Dynamics: Implications for Nuclear Architecture and Gene Expression  

NSDL National Science Digital Library

Studies of nuclear architecture reveal that the dynamic properties of proteins in the nucleus are critical for their function. The high mobility of proteins ensures their availability throughout the nucleus; their dynamic interplay generates an ever-changing, but overall stable, architectural framework, within which nuclear processes take place. As a consequence, overall nuclear morphology is determined by the functional interactions of nuclear components. The observed dynamic properties of nuclear proteins are consistent with a central role for stochastic mechanisms in gene expression and nuclear architecture.

Tom Mistelli (National Cancer Institute; )

2001-02-02

6

Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation  

Microsoft Academic Search

The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic

C Gerner; J Gotzmann; U Fröhwein; C Schamberger; A Ellinger; G Sauermann

2002-01-01

7

SiRNA screening of nuclear proteins.  

PubMed

Functional gene screening is a basic strategy for identifying candidate genes that are responsible for biological processes of interest to researchers. RNA-mediated interference (RNAi)-based screening is one such approach. Although the knockdown technique using siRNA is widely accepted, careful evaluation is required to avoid false positive candidates as much as possible, because various cellular responses can be caused by events other than the target gene disruption. In this chapter, we introduce a method for a relatively small-scale siRNA screening that is capable of finding RNA binding proteins that regulate a specific RNA that is a structural component of a nuclear compartment. PMID:25555592

Hasegawa, Yuko; Nakagawa, Shinichi

2015-01-01

8

Inner nuclear membrane proteins: targeting and influence on genome organization   

E-print Network

The nuclear envelope is a complex double membrane system that separates the activities of the nuclear and cytoplasmic compartments. A recent explosion in the number of proteins associated with this subnuclear organelle together ...

Zuleger, Nikolaj

2012-06-22

9

A complex of nuclear pore proteins required for pore function  

PubMed Central

A family of proteins bearing novel N-acetylglucosamine residues has previously been found to be required to form functional nuclear pores. To begin to determine which of the proteins in this family are essential for pore function, antisera were raised to each of three members of the family, p62, p58, and p54. With these antisera, it was possible to deplete nuclear reconstitution extracts of the proteins and to test the depleted nuclei for nuclear transport. In the course of the experiments, it was found that the three proteins exist as a complex; antisera to any one, while specific on a protein blot, coimmunoprecipitated all three proteins. This complex of pore proteins is stable to 2 M salt, 2 M urea, and the detergent Mega 10, indicating the presence of specific and tight protein-protein interactions. By gel filtration, the complex has a molecular mass of 550-600 kD. Nuclei containing pores depleted of the complex are found to be defective for nuclear transport; moreover, we observe a strict linear correlation between the amount of complex present in nuclei and the amount of nuclear transport of which those nuclei are capable. Thus, the p62-p58- p54 complex defines a group of proteins with strong protein-protein interactions that form a unit of pore structure essential for pore function. PMID:2050741

1991-01-01

10

The nuclear envelope LEM-domain protein emerin  

PubMed Central

Emerin, a conserved LEM-domain protein, is among the few nuclear membrane proteins for which extensive basic knowledge—biochemistry, partners, functions, localizations, posttranslational regulation, roles in development and links to human disease—is available. This review summarizes emerin and its emerging roles in nuclear “lamina” structure, chromatin tethering, gene regulation, mitosis, nuclear assembly, development, signaling and mechano-transduction. We also highlight many open questions, exploration of which will be critical to understand how this intriguing nuclear membrane protein and its “family” influence the genome. PMID:23873439

Berk, Jason M; Tifft, Kathryn E; Wilson, Katherine L

2013-01-01

11

Nuclear export dynamics of RNA–protein complexes  

Microsoft Academic Search

The central dogma of molecular biology — DNA makes RNA makes proteins — is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export

David Grünwald; Michael Rout; Robert H. Singer

2011-01-01

12

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

Microsoft Academic Search

A cytoplasmically anchored mutant SV40 T antigen, FS T antigen, was shown previously to inter- fere specifically with the nuclear import of a heterolo- gous 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., E Appel, and E. Fanning. 1991. Mol. Cell. Biol.

Karen van Zee; Achim Dickmanns; Utz Fischer; Reinhard Ltihrmann; Ellen Fanning

1993-01-01

13

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

14

Nuclear g protein signaling: new tricks for old dogs.  

PubMed

: According to the standard model of G protein-coupled receptor (GPCR) signaling, GPCRs are localized to the cell membrane where they respond to extracellular signals. Stimulation of GPCRs leads to the activation of heterotrimeric G proteins and their intracellular signaling pathways. However, this model fails to accommodate GPCRs, G proteins, and their downstream effectors that are found on the nuclear membrane or in the nucleus. Evidence from isolated nuclei indicates the presence of GPCRs on the nuclear membrane that can activate similar G protein-dependent signaling pathways in the nucleus as at the cell surface. These pathways also include activation of cyclic adenosine monophosphate, calcium and nitric oxide synthase signaling in cardiomyocytes. In addition, a number of distinct heterotrimeric and monomeric G proteins have been found in the nucleus of various cell types. This review will focus on understanding the function of nuclear G proteins with a focus on cardiac signaling where applicable. PMID:25590750

Campden, Rhiannon; Audet, Nicolas; Hébert, Terence E

2015-02-01

15

A Method for the Large-Scale Cloning of Nuclear Proteins and Nuclear Targeting Sequences on a Functional Basis  

Microsoft Academic Search

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after

Bruno Pichon; Dany Mercan; Valérie Pouillon; Christiane Christophe-Hobertus; Daniel Christophe

2000-01-01

16

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

17

Protein quality control at the inner nuclear membrane.  

PubMed

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O; Knop, Michael

2014-12-18

18

Nuclear envelope transmembrane proteins in differentiation systems   

E-print Network

Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA ...

Batrakou, Dzmitry G.

2012-11-30

19

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins  

PubMed Central

ABSTRACT Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins. PMID:25078698

Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra

2014-01-01

20

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

21

Extraction of nuclear proteins from root meristematic cells.  

PubMed

Fractionation and extraction of nuclear proteins are techniques intended to facilitate dedicated plant proteomic studies. These techniques rely on subcellular fractionation, which makes it possible to define and characterize the proteome of a subcellular organelle, in this case the cell nucleus. Nuclear protein fractionation is proposed as a method to be carried out according to the solubility of proteins in buffers of increasing ionic strength. This physical criterion, accompanied in some steps by the use of additional reagents, such as detergents or enzymes, produces fractions that have been demonstrated to have functional significance. The proposed procedure yields five fractions, the first of them containing proteins associated with the nuclear envelope and remnants of the cytoskeleton. The second fraction, which is soluble at low ionic strength, contains ribonucleoproteins active in nuclear RNA metabolism. After increasing ionic strength and digesting with DNase, the result is the chromatin fraction. Finally, the fourth and fifth fractions correspond to the nuclear matrix and are obtained, respectively, by solubilization in high salt concentration and in the form of the residual pellet, which is only soluble in 7 M urea under sonication. This procedure offers a wide range of applicability, even in the cases in which the genome of the particular species investigated is not sequenced. In general, the functional criteria driving the extraction method described here will make this method capable of generating valuable and useful information. PMID:17093303

González-Camacho, Fernando; Medina, Francisco Javier

2007-01-01

22

LIM-domain-only proteins: multifunctional nuclear transcription coregulators that interacts with diverse proteins.  

PubMed

The LIM-only subclass of LIM proteins is a family of nuclear transcription co-regulators that are characterized by the exclusive presence of two tandem LIM domains and no other functional domains. To date, four LIM-domain-only (LMO) proteins (LMO1-LMO4) have been identified. They regulate gene transcription by functioning as "linker" or "scaffolding" proteins with a remarkable potential to mediate protein-protein interactions. These proteins play important roles in cell fate determination, cell growth and differentiation, tissues patterning, and organ development. In this review, we briefly described the functions of LMO proteins in the organ development and diseases. We also summarized the interaction proteins of each LMO family member, which may contribute to elucidating the functions of these mysterious and important linker proteins. PMID:24379077

Sang, Meixiang; Ma, Li; Sang, Meijie; Zhou, Xinliang; Gao, Wei; Geng, Cuizhi

2014-02-01

23

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

24

Host Cell Nuclear Proteins Are Recruited to Cytoplasmic Vaccinia Virus Replication Complexes  

Microsoft Academic Search

The initiation and termination of vaccinia virus postreplicative transcription have been reported to require cellular proteins, some of which are believed to be nuclear proteins. Vaccinia virus replicates in the cytoplasmic compartment of the cell, raising questions as to whether vaccinia virus has access to nuclear proteins. This was addressed here by following the fate of several nuclear proteins after

Jaewook Oh; Steven S. Broyles

2005-01-01

25

Feline immunodeficiency virus Gag is a nuclear shuttling protein.  

PubMed

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus. PMID:22623802

Kemler, Iris; Saenz, Dyana; Poeschla, Eric

2012-08-01

26

Molecular chaperone-mediated nuclear protein dynamics.  

PubMed

Homeostasis requires effective action of numerous biological pathways including those working along a genome. The variety of processes functioning in the nucleus is considerable, yet the number of employed factors eclipses this total. Ideally, individual components assemble into distinct complexes and serially operate along a pathway to perform work. Adding to the complexity is a multitude of fluctuating internal and external signals that must be monitored to initiate, continue or halt individual activities. While cooperative interactions between proteins of the same process provide a mechanism for rapid and precise assembly, the inherent stability of such organized structures interferes with the proper timing of biological events. Further prolonging the longevity of biological complexes are crowding effects resulting from the high concentration of intracellular macromolecules. Hence, accessory proteins are required to destabilize the various assemblies to efficiently transition between structures, avoid off-pathway competitive interactions, and to terminate pathway activity. We suggest that molecular chaperones have evolved, in part, to manage these challenges by fostering a general and continuous dynamic protein environment within the nucleus. PMID:24694369

Echtenkamp, Frank J; Freeman, Brian C

2014-05-01

27

In vitro nuclear interactome of the HIV-1 Tat protein  

PubMed Central

Background One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. Results Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. Conclusion We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation. PMID:19454010

Gautier, Virginie W; Gu, Lili; O'Donoghue, Niaobh; Pennington, Stephen; Sheehy, Noreen; Hall, William W

2009-01-01

28

Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro  

SciTech Connect

Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

2003-06-11

29

A nuclear ubiquitin-proteasomal pathway targets inner nuclear membrane protein Asi2 for degradation  

PubMed Central

The nuclear envelope consists of inner and outer nuclear membranes. While the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane represents a unique membranous environment containing specific proteins. The mechanisms of integral inner nuclear membrane protein degradation are unknown. Here we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome and independent of the vacuole exhibiting a half-life of ? 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistently, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus. PMID:24928896

Boban, Mirta; Pantazopoulou, Marina; Schick, Anna; Ljungdahl, Per O.; Foisner, Roland

2015-01-01

30

Nuclear and nucleolar targeting of human ribosomal protein S6.  

PubMed Central

Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

Schmidt, C; Lipsius, E; Kruppa, J

1995-01-01

31

Nuclear Membrane Dynamics and Reassembly in Living Cells: Targeting of an Inner Nuclear Membrane Protein in Interphase and Mitosis  

Microsoft Academic Search

The mechanisms of localization and reten- tion of membrane proteins in the inner nuclear mem- brane and the fate of this membrane system during mi- tosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR-GFP). Photobleaching tech- niques revealed the majority of LBR-GFP to be com- pletely immobilized in

Jan Ellenberg; Eric D. Siggia; Jorge E. Moreira; Carolyn L. Smith; John F. Presley; Howard J. Worman; Jennifer Lippincott-Schwartz

1997-01-01

32

Regulation of Nuclear Localization of Signaling Proteins by Cytokinin  

SciTech Connect

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

Kieber, J.J.

2010-05-01

33

Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.  

PubMed Central

We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes. Images PMID:2583082

Lam, E; Green, P J; Wong, M; Chua, N H

1989-01-01

34

Sperm Nuclear Basic Proteins of Two Closely Related Species of Scorpaeniform Fish  

E-print Network

Sperm Nuclear Basic Proteins of Two Closely Related Species of Scorpaeniform Fish (Sebastes maliger of sperm nuclear basic proteins (SNBPs) in teleost fish. The distribution of the three basic groups­287, 2006. r 2006 Wiley-Liss, Inc. SPERM NUCLEAR BASIC PROTEINS FROM FISH From the very early attempts

Eirin Lopez, Jose Maria

35

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2?protein  

PubMed Central

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5? end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAP?, specifically bound VirD2 in vivo and in vitro. VirD2–AtKAP? interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAP? was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin ? family. Indeed, AtKAP? efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAP? specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells. PMID:9380702

Ballas, Nurit; Citovsky, Vitaly

1997-01-01

36

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; Ptá?ek, Louis; Fu, Ying-Hui

2014-01-01

37

p95vav associates with the nuclear protein Ku-70.  

PubMed Central

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed. PMID:8524317

Romero, F; Dargemont, C; Pozo, F; Reeves, W H; Camonis, J; Gisselbrecht, S; Fischer, S

1996-01-01

38

A method for dynamic nuclear polarization enhancement of membrane proteins.  

PubMed

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4 . Specifically, DNP MAS ssNMR experiments at 600?MHz/395?GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins. PMID:25504310

Smith, Adam N; Caporini, Marc A; Fanucci, Gail E; Long, Joanna R

2015-01-26

39

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

40

The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate  

PubMed Central

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases. PMID:24116158

Santos, Mariana; Rebelo, Sandra; Van Kleeff, Paula J. M.; Kim, Connie E.; Dauer, William T.; Fardilha, Margarida; da Cruz e Silva, Odete A.; da Cruz e Silva, Edgar F.

2013-01-01

41

Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases.  

PubMed Central

Decreased affinity of the retinoblastoma protein (RB) for the nuclear compartment has been correlated with cell cycle-dependent phosphorylation of the RB protein during the G1/S phase of the cell cycle. We examined the effects of microinjected protein-serine/threonine phosphatases types 1 (PP1) and 2A (PP2A) on nuclear association of RB monitored as the resistance of RB to extraction at the G1/S transition. Microinjection of PP1 into either the nucleus or the cytoplasm of cells synchronized in G1 increased the amount of RB that was resistant to extraction from the nucleus. Microinjection of PP2A, however, required direct injection into the nucleus to generate this effect. In addition, we found that nuclear injection of only the PP2A catalytic subunit (PP2AC) and not the complex containing the A and C subunits inhibited RB extraction. Microinjection of either PP1 or PP2A and the resultant increased affinity of RB for the nucleus corresponded with the inhibition of cell cycle progression into S phase. Injection of either phosphatase into cells that had entered S phase did not block DNA synthesis, suggesting that the effect of the injected phosphatases on cell cycle arrest was specific. In vitro biochemical studies with purified PP1 and PP2A showed that intact RB protein phosphorylated by cdc2 kinase served as a substrate for both protein phosphatases. Our results suggest that protein phosphatases may be important regulators of RB function and support the idea that cell cycle progression is regulated by the phosphorylation state of the RB protein. Images PMID:8380637

Alberts, A S; Thorburn, A M; Shenolikar, S; Mumby, M C; Feramisco, J R

1993-01-01

42

Mitogen activated protein kinase at the nuclear pore complex  

PubMed Central

Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

2011-01-01

43

Bacterial Delivery of Nuclear Proteins into Pluripotent and Differentiated Cells  

PubMed Central

Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming. PMID:21304583

Bichsel, Candace; Neeld, Dennis K.; Hamazaki, Takashi; Wu, Donghai; Chang, Lung-Ji; Yang, Lijun; Terada, Naohiro; Jin, Shouguang

2011-01-01

44

NLS-tagging: an alternative strategy to tag nuclear proteins  

PubMed Central

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.

2014-01-01

45

Nuclear targeting of the maize R protein requires two nuclear localization sequences  

SciTech Connect

Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding [beta]-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is found in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type [alpha]2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed. 45 refs., 6 figs.

Shieh, M.W.; Raikhel, N.V. (Michigan State Univ., East Lansing (United States)); Wessler, S.R. (Univ. of Georgia, Athens (United States))

1993-02-01

46

Dynamics of protein kinases: insights from nuclear magnetic resonance.  

PubMed

Protein kinases are ubiquitous enzymes with critical roles in cellular processes and pathology. As a result, researchers have studied their activity and regulatory mechanisms extensively. Thousands of X-ray structures give snapshots of the architectures of protein kinases in various states of activation and ligand binding. However, the extent of and manner by which protein motions and conformational dynamics underlie the function and regulation of these important enzymes is not well understood. Nuclear magnetic resonance (NMR) methods provide complementary information about protein conformation and dynamics in solution. However, until recently, the large size of these enzymes prevented researchers from using these methods with kinases. Developments in transverse relaxation-optimized spectroscopy (TROSY)-based techniques and more efficient isotope labeling strategies are now allowing researchers to carry out NMR studies on full-length protein kinases. In this Account, we describe recent insights into the role of dynamics in protein kinase regulation and catalysis that have been gained from NMR measurements of chemical shift changes and line broadening, residual dipolar couplings, and relaxation. These findings show strong associations between protein motion and events that control kinase activity. Dynamic and conformational changes occurring at ligand binding sites and other regulatory domains of these proteins propagate to conserved kinase core regions that mediate catalytic function. NMR measurements of slow time scale (microsecond to millisecond) motions also reveal that kinases carry out global exchange processes that synchronize multiple residues and allosteric interconversion between conformational states. Activating covalent modifications or ligand binding to form the Michaelis complex can induce these global processes. Inhibitors can also exploit the exchange properties of kinases by using conformational selection to form dynamically quenched states. These investigations have revealed that kinases are highly dynamic enzymes, whose regulation by interdomain interactions, ligand binding, and covalent modifications involve changes in motion and conformational equilibrium in a manner that can be correlated with function. Thus, NMR provides a unique window into the role of protein dynamics in kinase regulation and catalysis with important implications for drug design. PMID:25803188

Xiao, Yao; Liddle, Jennifer C; Pardi, Arthur; Ahn, Natalie G

2015-04-21

47

Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bbeta.  

PubMed

Protein kinase B (PKB) is a member of the second messenger-dependent family of serine/threonine kinases that has been implicated in signaling pathways downstream of growth factor receptor tyrosine kinases and phosphatidylinositol 3-kinase. Here we report the characterization of the human beta-isoform of PKB (PKBbeta). PKBbeta is ubiquitously expressed in a number of human tissues, with mRNA and protein levels elevated in heart, liver, skeletal muscle, and kidney. After transfection into HEK-293 or COS-1 cells, PKBbeta is activated 2- to 12-fold by mitogens and survival factors. Activation was due to phosphorylation on Thr-309 and Ser-474, which correspond to Thr-308 and Ser-473 implicated in the regulation of PKBalpha. Both phosphorylation and activation were prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Moreover, membrane-targeted PKBbeta was constitutively activated when overexpressed in HEK-293 cells. Although the specific activity of PKBbeta was lower than that of PKBalpha toward Crosstide as a substrate (23 nmol/min/mg compared with 178 nmol/min/mg for PKBalpha), both enzymes showed similar substrate specificities. Using confocal microscopy, we show that activation of PKBbeta results in its nuclear translocation within 20 to 30 min after stimulation. These observations provide evidence that PKBbeta undergoes nuclear translocation upon mitogenic activation and support a role for PKB in signaling from receptor tyrosine kinases to the nucleus through phosphatidylinositol 3-kinase. PMID:9374542

Meier, R; Alessi, D R; Cron, P; Andjelkovi?, M; Hemmings, B A

1997-11-28

48

Switch from capsid protein import to adenovirus assembly by cleavage of nuclear transport signals  

PubMed Central

Replication and assembly of adenovirus occurs in the nucleus of infected cells, requiring the nuclear import of all viral structural proteins. In this report we show that nuclear import of the major capsid protein, hexon, is mediated by protein VI, a structural protein located underneath the 12 vertices of the adenoviral capsid. Our data indicate that protein VI shuttles between the nucleus and the cytoplasm and that it links hexon to the nuclear import machinery via an importin ?/?-dependent mechanism. Key nuclear import and export signals of protein VI are located in a short C-terminal segment, which is proteolytically removed during virus maturation. The removal of these C-terminal transport signals appears to trigger a functional transition in protein VI, from a role in supporting hexon nuclear import to a structural role in virus assembly. PMID:14633984

Wodrich, Harald; Guan, Tinglu; Cingolani, Gino; Von Seggern, Dan; Nemerow, Glen; Gerace, Larry

2003-01-01

49

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

Microsoft Academic Search

Heterogeneous nuclear RNAs and specific nuclear proteins form heterogeneous nuclear ribonucleoprotein complexes (hnRNPs), one of the most abundant components of the nucleus. In mitosis, as the nuclear envelope breaks down, hnRNPs disperse throughout the cell. At the end of mitosis, hnRNPs dissociate and their proteins are transported into the daughter cell nuclei separately. Some are transported immediately (early group), while

Serafin Pinol-Roma; Gideon Dreyfuss

1991-01-01

50

An analysis of the protein interactions involved in classical nuclear import  

E-print Network

Every aspect of eukaryotic cellular life involves the trafficking of proteins and other biomolecules through the nuclear membrane for the purposes of bringing feedback to or information from the nucleus. Many proteins larger than 40 kDa must...

Reedy, Brian Michael Thomas

1999-01-01

51

Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*  

PubMed Central

Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

2011-01-01

52

Insulin increases nuclear protein kinase Cdelta in L6 skeletal muscle cells.  

PubMed

Protein kinase C (PKC) isoforms are involved in the transduction of a number of signals important for the regulation of cell growth, differentiation, apoptosis, and other cellular functions. PKC proteins reside in the cytoplasm in an inactive state translocate to various membranes to become fully activated in the presence of specific cofactors. Recent evidence indicates that PKC isoforms have an important role in the nucleus. We recently showed that insulin rapidly increases PKCdelta RNA and protein. In this study we initially found that insulin induces an increase in PKCdelta protein in the nuclear fraction. We therefore attempted to elucidate the mechanism of the insulin-induced increase in nuclear PKCdelta. Studies were performed on L6 skeletal myoblasts and myotubes. The increase in nuclear PKCdelta appeared to be unique to insulin because it was not induced by other growth factors or rosiglitazone. Inhibition of transcription or translation blocked the insulin-induced increase in nuclear PKCdelta, whereas inhibition of protein import did not. Inhibition of protein export from the nucleus reduced the insulin-induced increase in PKCdelta in the cytoplasm and increased it in the nucleus. The increase in nuclear PKCdelta induced by insulin was reduced but not abrogated by treatment of isolated nuclei by trypsin digestion. Finally, we showed that insulin induced incorporation of (35)S-methionine into nuclear PKCdelta protein; this effect was not blocked by inhibition of nuclear import. Thus, these results suggest that insulin may induce nuclear-associated, or possibly nuclear, translation of PKCdelta protein. PMID:18162512

Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Kesten, Dov; Sampson, Sanford R

2008-04-01

53

Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery  

PubMed Central

Background Different cell types have distinctive patterns of chromosome positioning in the nucleus. Although ectopic affinity-tethering of specific loci can be used to relocate chromosomes to the nuclear periphery, endogenous nuclear envelope proteins that control such a mechanism in mammalian cells have yet to be widely identified. Results To search for such proteins, 23 nuclear envelope transmembrane proteins were screened for their ability to promote peripheral localization of human chromosomes in HT1080 fibroblasts. Five of these proteins had strong effects on chromosome 5, but individual proteins affected different subsets of chromosomes. The repositioning effects were reversible and the proteins with effects all exhibited highly tissue-restricted patterns of expression. Depletion of two nuclear envelope transmembrane proteins that were preferentially expressed in liver each reduced the normal peripheral positioning of chromosome 5 in liver cells. Conclusions The discovery of nuclear envelope transmembrane proteins that can modulate chromosome position and have restricted patterns of expression may enable dissection of the functional relevance of tissue-specific patterns of radial chromosome positioning. PMID:23414781

2013-01-01

54

Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan)] [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)] [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan)] [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)] [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

2012-06-29

55

Dynamics and diverse functions of nuclear pore complex proteins  

PubMed Central

Nuclear pore complexes (NPCs) are best known for their central role in controlling the molecular trafficking between the cytoplasm and the nucleus. NPCs are assembled from about 30 different proteins and a growing body of evidence suggests that these nucleoporins are not only acting in the context of NPCs, but also in the nucleoplasm and cytoplasm. In this context it is well accepted that a set of nucleoporins are important regulators of a variety of mitotic processes, including kinetochore assembly, spindle checkpoint control and cytokinesis, whereas others associate with chromatin and administer gene expression. However, the functional importance of nucleoporins go far beyond these roles and this review will provide an overview of the latest insights into the versatility of metazoan nucleoporins with an emphasis on their roles in cell migration, cellular signaling and tissue-specific activities. PMID:22555605

Chatel, Guillaume; Fahrenkrog, Birthe

2012-01-01

56

Inner nuclear membrane protein LEM-2 is required for correct nuclear separation and morphology in C. elegans.  

PubMed

The inner nuclear membrane proteins emerin and LEMD2 have both overlapping and separate functions in regulation of nuclear organization, gene expression and cell differentiation. We report here that emerin (EMR-1) and LEM domain protein 2 (LEM-2) are expressed in all tissues throughout Caenorhaditis elegans development but their relative distribution differs between cell types. The ratio of EMR-1 to LEM-2 is particularly high in contractile tissues, intermediate in neurons and hypodermis and lowest in intestine and germ line. We find that LEM-2 is recruited earlier than EMR-1 to reforming nuclear envelopes, suggesting the presence of separate mitotic membrane compartments and specific functions of each protein. Concordantly, we observe that nuclei of lem-2 mutant embryos, but not of emr-1 mutants, have reduced nuclear circularity. Finally, we uncover a so-far-unknown role of LEM-2 in nuclear separation and anchoring of microtubule organizing centers. PMID:25653391

Morales-Martínez, Adela; Dobrzynska, Agnieszka; Askjaer, Peter

2015-03-15

57

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

58

Regulation of Greatwall kinase by protein stabilization and nuclear localization.  

PubMed

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin ? and ? families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry. PMID:25483093

Yamamoto, Tomomi M; Wang, Ling; Fisher, Laura A; Eckerdt, Frank D; Peng, Aimin

2014-01-01

59

TATA-binding protein and nuclear differentiation in Tetrahymena thermophila.  

PubMed Central

Unambiguous TATA boxes have not been identified in upstream sequences of Tetrahymena thermophila genes analyzed to date. To begin a characterization of the promoter requirements for RNA polymerase II, the gene encoding TATA-binding protein (TBP) was cloned from this species. The derived amino acid sequence for the conserved C-terminal domain of Tetrahymena TBP is one of the most divergent described and includes a unique 20-amino-acid C-terminal extension. Polyclonal antibodies generated against a fragment of Tetrahymena TBP recognize a 36-kDa protein in macronuclear preparations and also cross-react with yeast and human TBPs. Immunocytochemistry was used to examine the nuclear localization of TBP during growth, starvation, and conjugation (the sexual phase of the life cycle). The transcriptionally active macronuclei stained at all stages of the life cycle. The transcriptionally inert micronuclei did not stain during growth or starvation but surprisingly stained with anti-TBP throughout early stages of conjugation. Anti-TBP staining disappeared from developing micronuclei late in conjugation, corresponding to the onset of transcription in developing macronuclei. Since micronuclei do not enlarge or divide at this time, loss of TBP appears to be an active process. Thus, the transcriptional differences between macro- and micronuclei that arise during conjugation are associated with the loss of a major component of the basal transcription apparatus from developing micronuclei rather than its appearance in developing macronuclei. Images PMID:8264641

Stargell, L A; Gorovsky, M A

1994-01-01

60

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

61

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

62

Examining the mechanism of Erk nuclear translocation using green fluorescent protein  

Microsoft Academic Search

In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled

Angela M Horgan; Philip J. S Stork

2003-01-01

63

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

E-print Network

Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas Dennis D and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene, and immunocytochemical studies dem- onstrate that this protein is nuclear-localized under both nutrient- replete

64

HIN-200: a novel family of IFN-inducible nuclear proteins  

Microsoft Academic Search

In recent years a novel family of inter- feron (IFN)-inducible nuclear proteins has been identified in both mouse and humans and implicated in the control of myeloid differentiation. Recent evidence suggests that this role may be mediated by an effect on cell cycle regulation through interaction with known transcriptional regulators. Our labora- tory first identified a nuclear protein designated IF!

Michelle J. Dawson; Joseph A. Trapani

65

The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals  

Microsoft Academic Search

Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in

Derek E. Eberhart; Henry E. Malter; Yue Feng; Stephen T. Warren

1996-01-01

66

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. Promyelocytic leukemia nuclear bodies are implicated in important regulatory processes. To understand leukemia nuclear bodies accurately when interaction data is available. At a false positive rate of 10

Dellaire, Graham

67

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

68

The nuclear import of ribosomal proteins is regulated by mTOR  

PubMed Central

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.

2014-01-01

69

Protein Interactions Involved in Nuclear Import of the Agrobacterium VirE2 Protein in Vivo and in Vitro*  

E-print Network

, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformationProtein Interactions Involved in Nuclear Import of the Agrobacterium VirE2 Protein in Vivo

Citovsky, Vitaly

70

Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.  

PubMed

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions. PMID:8876198

Jurata, L W; Kenny, D A; Gill, G N

1996-10-15

71

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

72

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

73

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein  

SciTech Connect

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.

Inagaki, Yuichi [Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812 (Japan); Mitsutake, Susumu [Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812 (Japan); Igarashi, Yasuyuki [Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812 (Japan)]. E-mail: yigarash@pharm.hokudai.ac.jp

2006-05-12

74

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

75

A novel cell-cycle-dependent 350-kDa nuclear protein: C-terminal domain sufficient for nuclear localization.  

PubMed

We have screened human scleroderma patients for immunoreactivity with the components of the nucleus and the mitotic apparatus. We announce the identification of a novel cell-cycle-dependent nuclear protein using serum from a CREST patient AH. AH protein first appears at the nucleus of G2-phase and associates with the centrosome throughout the cell cycle. As chromosomes condense during the prophase, AH protein becomes enriched at the kinetochores. During mitosis, AH protein progressively disperses from the kinetochore and becomes diffusely localized in the cytoplasm and in telophase; it appears to be enriched within the intracellular bridge. Molecular cloning and transfection studies reveal that the 350-kDa AH protein contains a coiled-coil and a globular domain at the C-terminus that is sufficient for nuclear localization. PMID:7612011

Li, Q; Ke, Y; Kapp, J A; Fertig, N; Medsger, T A; Joshi, H C

1995-07-01

76

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

PubMed Central

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

2014-01-01

77

Fate of the Inner Nuclear Membrane Protein Lamin B Receptor and Nuclear Lamins in Herpes Simplex Virus Type 1 Infection  

PubMed Central

During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B2 tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B1, and B2. By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane. PMID:11507226

Scott, Emily S.; O'Hare, Peter

2001-01-01

78

ANP32B Is a Nuclear Target of Henipavirus M Proteins  

PubMed Central

Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed. PMID:24823948

Bauer, Anja; Neumann, Sebastian; Karger, Axel; Henning, Ann-Kristin; Maisner, Andrea; Lamp, Boris; Dietzel, Erik; Kwasnitschka, Linda; Balkema-Buschmann, Anne; Keil, Günther M.; Finke, Stefan

2014-01-01

79

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

80

Identification of Protein-Bound Dinitrosyl Iron Complexes by Nuclear Resonance Vibrational Spectroscopy  

E-print Network

We have applied [superscript 57]Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N?Fe?N unit can be observed between 500 ...

Wang, Hongxin

81

Nuclear Magnetic Resonance based Characterization of the Protein Binding Pocket using Hyperpolarized Ligand  

E-print Network

Polarization (DNP) combined with Nuclear Magnetic Resonance (NMR) has emerged as a new tool for studying interactions between different molecules. In this study, the DNP-NMR technique was employed for characterization of the protein binding pocket through...

Min, Hlaing

2014-08-04

82

Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences  

E-print Network

LETTERS Arthropod relationships revealed by phylogenomic analysis of nuclear protein , Regina Wetzer5 , Joel W. Martin5 & Clifford W. Cunningham4 The remarkable antiquity, diversity and ecological significance of arthropods have inspired numerous attempts to resolve their deep phylogenetic

Cunningham, Cliff

83

NEX-TRAP, a novel method for in vivo analysis of nuclear export of proteins.  

PubMed

Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin ?- and ?-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remain tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): a potential nuclear export cargo is fused to FRB, to EYFP for direct visualization as well as to an SV40-derived nuclear localization signal (NLS) for constitutive nuclear import. An integral membrane protein that resides at the trans Golgi network (TGN) is fused to a cytoplasmically exposed FKBP and serves as reporter. EYFP-NLS-FRB fusion proteins with export activity accumulate in the nucleus at steady state but continuously shuttle between nucleus and cytoplasm. Rapamycin-induced dimerization of FRB and FKBP at the TGN traps the shuttling protein outside of the nucleus, making nuclear export permanent. Using several example cargoes, we show that the NEX-TRAP is superior to existing assays owing to its ease of use, its sensitivity and accuracy. Analysis of large numbers of export cargoes is facilitated by recombinational cloning. The NEX-TRAP holds the promise of applicability in automated fluorescence imaging for systematic analysis of nuclear export, thereby improving in silico prediction of nuclear export sequences. PMID:22708827

Raschbichler, Verena; Lieber, Diana; Bailer, Susanne M

2012-10-01

84

A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts  

Microsoft Academic Search

Immediately after the initiation of transcrip- tion in eukaryotes, nascent RNA polymerase II tran- scripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, hnRNP complexes from HeLa cell nuclei contain >20 major proteins in the molecu- lar mass range of 34,000-120,000 D. Among these are the previously described A, B, and C groups

S. Pinol-Roma; Maurice S. Swanson; Joseph G. Gall; Gideon Dreyfuss

1989-01-01

85

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

86

High-Frequency Dynamic Nuclear Polarization in MAS Spectra of Membrane and Soluble Proteins  

E-print Network

High-Frequency Dynamic Nuclear Polarization in MAS Spectra of Membrane and Soluble Proteins Melanie The success of these experiments is due largely to the ability to record multidimensional MAS NMR spectra this sensitivity problem, we have recently developed high-field dynamic nuclear polarization (DNP) experiments

Griffin, Robert G.

87

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

88

Expression of the Nuclear Receptor Coactivator, cAMP Response Element-Binding Protein, Is Sexually  

E-print Network

. Acetylation of histones by coactivators removes a pos- itive charge, leading to a reduction in higherExpression of the Nuclear Receptor Coactivator, cAMP Response Element-Binding Protein, Is Sexually found that on the day of birth (postnatal d 0) males express higher levels of the nuclear receptor

89

Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein  

SciTech Connect

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.

Au, Victoria; Yu Mei [Department of Microbiology and Immunology, Queen's University, Kingston, ON, K7L 3N6 (Canada); Carstens, Eric B. [Department of Microbiology and Immunology, Queen's University, Kingston, ON, K7L 3N6 (Canada)], E-mail: Carstens@queensu.ca

2009-03-01

90

Evidence for Ubiquitin-Regulated Nuclear and Subnuclear Trafficking among Paramyxovirinae Matrix Proteins.  

PubMed

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype. PMID:25782006

Pentecost, Mickey; Vashisht, Ajay A; Lester, Talia; Voros, Tim; Beaty, Shannon M; Park, Arnold; Wang, Yao E; Yun, Tatyana E; Freiberg, Alexander N; Wohlschlegel, James A; Lee, Benhur

2015-02-01

91

Evidence for Ubiquitin-Regulated Nuclear and Subnuclear Trafficking among Paramyxovirinae Matrix Proteins  

PubMed Central

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype. PMID:25782006

Pentecost, Mickey; Vashisht, Ajay A.; Beaty, Shannon M.; Park, Arnold; Wang, Yao E.; Yun, Tatyana E; Freiberg, Alexander N.; Wohlschlegel, James A.; Lee, Benhur

2015-01-01

92

DNA-protein interactions between mammalian nuclear proteins and a GCC-element included in a composite cis-acting element of mouse ribosomal protein L32 promoter.  

PubMed

DNA-protein complex formation between the sequence GC(GCC)4 (GCC-element) of mouse ribosomal protein L32 (rpL32) promoter and nuclear proteins of mouse and human cells has been studied using gel retardation and South-Western blotting methods. The rpL32 promoter fragment (-24.+11) was able to form specific complexes with mouse and human nuclear proteins mainly due to the presence of the GCC-element (-19.-6). DNA-protein complex patterns exhibited marked tissue-specificity. Three nuclear polypeptides of approximately 18, 28, and 50 kD that bind to the rpL32 promoter region (-24.+11) have been detected in HeLa cells by ligand blotting. At least one of them (18 kD) interacted with the GCC-element directly. The same fragment of the promoter interacted only with one nuclear polypeptide (28-31 kD) from human fibroblasts. DNA-protein complex formation between the investigated rpL32 promoter fragment containing the GCC-element and human fibroblast nuclear proteins is Zn2+-dependent. The method of functional titration (in vivo competition in the CAT-test) revealed that the GCC-element within the rpL32 promoter functions as a positive cis-acting transcriptional element in NIH 3T3 cells. Thus, our data characterize the sequence GC(GCC)4 as a functionally active cis-element included as a component in the more complex (composite) cis-element of mouse rpL32 promoter exhibiting tissue-specific properties. In various mammalian cell types the GCC-element can interact with various nuclear proteins, and the mode of these interactions can be determined by its relative position to other cis-elements in the regulatory sites of the genome. PMID:10187914

Orlov, S V; Kuteikin, K B; Dizhe, E B; Kuryshev, V Y; Shpakovich, V M; Perevozchikov, A P

1999-02-01

93

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

94

Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock  

Microsoft Academic Search

The Drosophila period protein (PER) is a predominantly nuclear protein and a likely component of a circadian clock. PER is required for daily oscillations in the transcription of its own gene and thus participates in a circadian feedback loop. In this study, key pacemaker neurons of the Drosophila brain were examined to determine whether the subcellular distribution of PER changes

Kathryn D Curtin; Zuoshi J Huang; Michael Rosbash

1995-01-01

95

A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus  

Microsoft Academic Search

The basic protein of Autographa californica nuclear polyhedrosis virus (AcMNPV) is associated with virus DNA in virion nucleocapsids and is produced in infected cells during the late phase of gene expression. A transfer vector was constructed containing the fl- galactosidase gene, under the control of a copy of the putative basic protein promoter, in place of the polyhedrin gene within

Michele S. Hill-Perkins; Robert D. Possee

1990-01-01

96

Sexual dimorphism of rat liver nuclear proteins: regulatory role of growth hormone.  

PubMed

Many genes are expressed in mammalian liver in a sexually dimorphic manner. DNA microarray analysis has shown that growth hormone (GH) and its sex-dependent pattern of pituitary secretion play a major role in establishing the sexually dimorphic patterns of liver gene expression. However, GH may exert effects on protein post-translational modification and nuclear localization that are not reflected at the mRNA level. To investigate these potential effects of GH, we used two-dimensional gel electrophoresis followed by LC-MS/MS to: 1) identify rat liver nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences; and 2) determine the role of the plasma GH profile in establishing these differences. Nuclear extracts prepared from livers of individual male (n=9) and female (n=5) adult rats, and from males given GH by continuous infusion for 7 days to feminize liver gene expression (n=5 rats), were resolved by two-dimensional electrophoresis. Image analysis of SYPRO Ruby-stained gels revealed 165 sexually dimorphic protein spots that differ in normalized volume between male and female groups by >1.5-fold at p<0.05. Sixty of these proteins exhibited female-like changes in spot abundance following continuous GH treatment. Comparison of male and GH-treated male groups revealed 130 proteins that displayed >1.5-fold differences in abundance, with 60 of these GH-responsive spots being sexually dimorphic. Thus, GH plays an important role in establishing the sex-dependent differences in liver nuclear protein content. Twenty-eight of the sexually dimorphic and/or GH-regulated protein spots were identified by LC-MS/MS. Proteins identified include regucalcin, nuclear factor 45, and heterogeneous nuclear ribonucleoproteins A3, D-like, and K, in addition to proteins such as GST, normally associated with cytosolic extracts but also reported to be localized in the nucleus. PMID:15456855

Laz, Ekaterina V; Wiwi, Christopher A; Waxman, David J

2004-12-01

97

A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3  

SciTech Connect

The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.

Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning [Department of Microbiology, Harbin Medical University, Harbin 150081 (China)] [Department of Microbiology, Harbin Medical University, Harbin 150081 (China); Zhao, Wenran, E-mail: wenran.zhao@gmail.com [Department of Cell Biology, Harbin Medical University, Harbin 150081 (China)] [Department of Cell Biology, Harbin Medical University, Harbin 150081 (China); Zhong, Zhaohua, E-mail: zhonghmu@gmail.com [Department of Microbiology, Harbin Medical University, Harbin 150081 (China)] [Department of Microbiology, Harbin Medical University, Harbin 150081 (China)

2012-11-25

98

The nuclear localization of SWI/SNF proteins is subjected to oxygen regulation  

PubMed Central

Background Hypoxia is associated with many disease conditions in humans, such as cancer, stroke and traumatic injuries. Hypoxia elicits broad molecular and cellular changes in diverse eukaryotes. Our recent studies suggest that one likely mechanism mediating such broad changes is through changes in the cellular localization of important regulatory proteins. Particularly, we have found that over 120 nuclear proteins with important functions ranging from transcriptional regulation to RNA processing exhibit altered cellular locations under hypoxia. In this report, we describe further experiments to identify and evaluate the role of nuclear protein relocalization in mediating hypoxia responses in yeast. Results To identify regulatory proteins that play a causal role in mediating hypoxia responses, we characterized the time courses of relocalization of hypoxia-altered nuclear proteins in response to hypoxia and reoxygenation. We found that 17 nuclear proteins relocalized in a significantly shorter time period in response to both hypoxia and reoxygenation. Particularly, several components of the SWI/SNF complex were fast responders, and analysis of gene expression data show that many targets of the SWI/SNF proteins are oxygen regulated. Furthermore, confocal fluorescent live cell imaging showed that over 95% of hypoxia-altered SWI/SNF proteins accumulated in the cytosol in hypoxic cells, while over 95% of the proteins were nuclear in normoxic cells, as expected. Conclusions SWI/SNF proteins relocalize in response to hypoxia and reoxygenation in a quick manner, and their relocalization likely accounts for, in part or in whole, oxygen regulation of many SWI/SNF target genes. PMID:22932476

2012-01-01

99

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

PubMed Central

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

2014-01-01

100

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

101

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.  

PubMed Central

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor. Images PMID:2583097

Weber, K; Plessmann, U; Ulrich, W

1989-01-01

102

Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions.  

PubMed Central

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions. Images PMID:1825347

Fresco, L D; Harper, D S; Keene, J D

1991-01-01

103

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

104

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

105

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

106

Role of Herpes Simplex Virus 1 Immediate Early Protein ICP22 in Viral Nuclear Egress  

PubMed Central

ABSTRACT In order to investigate the novel function(s) of the herpes simplex virus 1 (HSV-1) immediate early protein ICP22, we screened for ICP22-binding proteins in HSV-1-infected cells. Our results were as follows. (i) Tandem affinity purification of ICP22 from infected cells, coupled with mass spectrometry-based proteomics and subsequent analyses, demonstrates that ICP22 forms a complex(es) with the HSV-1 proteins UL31, UL34, UL47 (or VP13/14), and/or Us3. All these proteins were previously reported to be important for viral egress through the nuclear membrane. (ii) ICP22 colocalizes with UL31 and UL34 at the nuclear membrane in wild-type HSV-1-infected cells. (iii) The UL31-null mutation prevents the targeting of ICP22 to the nuclear membrane. (iv) The ICP22-null mutation resulted in UL31 and UL34 being mislocalized in the endoplasmic reticulum (in addition to the nuclear membrane) and significantly reduced numbers of primary enveloped virions in the perinuclear space, although capsids accumulated in the nuclei. Collectively, these results suggest that (i) ICP22 interacts with HSV-1 regulators of nuclear egress, including UL31, UL34, UL47, and Us3 in HSV-1-infected cells; (ii) UL31 mediates the recruitment and anchorage of ICP22 at the nuclear membrane; and (iii) ICP22 plays a regulatory role in HSV-1 primary envelopment, probably by interacting with and regulating UL31 and UL34. Here we report a previously unknown function for ICP22 in the regulation of HSV-1 nuclear egress. IMPORTANCE The herpes simplex virus 1 (HSV-1) immediate early protein ICP22 is recognized primarily as a regulator of viral gene expression. In this study, we show that ICP22 interacts with the HSV-1 proteins UL31 and UL34, which play crucial roles at the nuclear membrane in HSV-1 primary envelopment during viral nuclear egress. We also demonstrate that UL31 is required for the targeting of ICP22 to the nuclear membrane and that ICP22 is required for the correct localization of UL31 and/or UL34. Furthermore, we confirm that ICP22 is required for efficient HSV-1 primary envelopment during viral nuclear egress. Thus, we report, for the first time, that ICP22 plays a regulatory role in HSV-1 nuclear egress. PMID:24741100

Maruzuru, Yuhei; Shindo, Keiko; Liu, Zhuoming; Oyama, Masaaki; Kozuka-Hata, Hiroko; Arii, Jun; Kato, Akihisa

2014-01-01

107

Characterizing fluorescence recovery curves for nuclear proteins undergoing binding events  

Microsoft Academic Search

Fluorescence recovery after photobleaching (FRAP) is an experimental technique used to measure the mobility of proteins within\\u000a the cell nucleus. After proteins of interest are fluorescently tagged for their visualization and monitoring, a small region\\u000a of the nucleus is photobleached. The experimental FRAP data are obtained by recording the recovery of the fluorescence in\\u000a this region over time.\\u000a \\u000a In this

G. Carrero; E. Crawford; M. J. Hendzel; G. de Vries

2004-01-01

108

Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.  

PubMed Central

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities. Images PMID:3386636

Swanson, M S; Dreyfuss, G

1988-01-01

109

A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast.  

PubMed

Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins. Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport. Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates. However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far. By the use of conditional yeast mutants, we examined nuclear protein import in vivo. We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction. In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected. Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import. In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking. PMID:12393908

Stade, Katrin; Vogel, Frank; Schwienhorst, Ingrid; Meusser, Birgit; Volkwein, Corinna; Nentwig, Brigitte; Dohmen, R Jürgen; Sommer, Thomas

2002-12-20

110

Nuclear Magnetic Resonance Structural Studies of Membrane Proteins in Micelles and Bilayers  

PubMed Central

Summary Nuclear magnetic resonance (NMR) spectroscopy enables determination of membrane protein structures in lipid environments, such as micelles and bilayers. This chapter outlines the steps for membrane-protein structure determination using solution NMR with micelle samples, and solid-state NMR with oriented lipid-bilayer samples. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from ? and CHIF, two membrane proteins that function as regulatory subunits of the Na+- and K+-ATPase. PMID:17951757

Gong, Xiao-Min; Franzin, Carla M.; Thai, Khang; Yu, Jinghua; Marassi, Francesca M.

2010-01-01

111

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

112

A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts  

PubMed Central

Immediately after the initiation of transcription in eukaryotes, nascent RNA polymerase II transcripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. hnRNP complexes from HeLa cell nuclei contain greater than 20 major proteins in the molecular mass range of 34,000- 120,000 D. Among these are the previously described A, B, and C groups of proteins (34,000-43,000 D) and several larger, and as yet uncharacterized, proteins. Here we describe the isolation and characterization of a novel hnRNP protein termed the L protein (64-68 kD by mobility in SDS-polyacrylamide gels). Although L is a bona fide component of hnRNP complexes, it also appears to be a different type of hnRNP protein from those previously characterized. A considerable amount of L is found outside hnRNP complexes, and monoclonal antibodies to the L protein also strongly stain unidentified discrete nonnucleolar structures, in addition to nucleoplasm, in HeLa cell nuclei. Interestingly, the same antibodies stain the majority of nonnucleolar nascent transcripts from the loops of lampbrush chromosomes in the newt, but the most intense staining is localized to the landmark giant loops. The L protein is the first protein of giant loops identified so far, and antibodies to it thus provide a useful tool with which to study these unique RNAs. In addition, isolation and sequencing of cDNA clones for the L protein from human cells predicts a glycine- and proline-rich protein of 60,187 D, which contains two 80 amino acid segments only distantly related to the RNP consensus sequence-type RNA- binding domain. The L protein, therefore, is a new type of hnRNP protein. PMID:2687284

1989-01-01

113

Studies in protein dynamics using heteronuclear nuclear magnetic resonance spectroscopy  

NASA Astrophysics Data System (ADS)

Dynamic processes in proteins are important for their biological function. Several issues in protein dynamics are addressed by applying existing NMR methodologies to investigate dynamics of several small proteins. Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1--56. The results suggest that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9. Single domain proteins can fold in vitro at rates in excess of 1 x 104 s-1. Measurement of folding rates of this magnitude poses a considerable technical challenge. Off-resonance 15N R1rho measurements are shown to be capable of measuring such fast protein folding rates. The measurements were performed on a sample of the peripheral subunit-binding domain from the dihydrolopoamide acetyltransferase component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus 15N labeled at Ala 11. Fast intramolecular motions (on ps-ns time scale) can be studied by heteronuclear laboratory frame NMR relaxation. The temperature dependence of the backbone dynamics of the 36-resiude subdomain of the F-actin bundling protein villin has been investigated by studying the temperature dependence of order parameters obtained from 15N relaxation measurements. The results support the hypothesis that one of the possible mechanisms of thermostability is to lower the heat capacity difference between the folded and unfolded states by lowering the contribution from the backbone dynamics. A commonly used model-free approach for the interpretation of the relaxation data for macromolecules in solution is modified to correct for the decoupling approximation between the overall and internal motions.

Vugmeyster, Liliya

114

CGGBP1 is a nuclear and midbody protein regulating abscission  

SciTech Connect

Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletion resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.

Singh, Umashankar, E-mail: umashankar.singh@genpat.uu.se; Westermark, Bengt

2011-01-15

115

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

116

Mutants in a yeast Ran binding protein are defective in nuclear transport.  

PubMed Central

Ran, a Ras-like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione-S-transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP-bound state. A major protein with the apparent size of 34 kDa co-purifies with the GTP-bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature-sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope. Images PMID:7489726

Schlenstedt, G; Wong, D H; Koepp, D M; Silver, P A

1995-01-01

117

Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants.  

PubMed

Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain-interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions. PMID:24891605

Zhou, Xiao; Graumann, Katja; Wirthmueller, Lennart; Jones, Jonathan D G; Meier, Iris

2014-06-01

118

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

119

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

PubMed Central

KAR5 is required for membrane fusion during 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 shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex. PMID:9382856

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

1997-01-01

120

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 (G?) 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 G?5 (R7-G?5)] shuttles between the plasma membrane and the nucleus with assistance from a novel shuttle protein, R7BP. R7BP binds directly to R7-G?5 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-G?5 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

121

A viral movement protein as a nuclear shuttle. The geminivirus BR1 movement protein contains domains essential for interaction with BL1 and nuclear localization.  

PubMed Central

For the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, BR1 and BL1. These act in a cooperative manner to transport the viral single-stranded DNA genome from its site of replication in the nucleus to the cell periphery (A.A. Sanderfoot, S.G. Lazarowitz [1995] Plant Cell 7: 1185-1194). We have proposed that BR1 functions as a nuclear shuttle protein, transporting the viral single-stranded DNA to and from the nucleus as a complex that is recognized by BL1 for movement to adjacent cells. To further investigate this, we expressed BR1 mutants known to affect viral infectivity in Spodoptera frugiperda insect cells and Nicotiana tabacum L. cv Xanthi protoplasts and found these to be defective in either their nuclear targeting or their ability to be redirected to the cell periphery when co-expressed with BL1. Translational fusions to beta-glucuronidase and alanine-scanning mutagenesis further demonstrated that the C-terminal 86 amino acids of BR1 contains a domain(s) essential for its interaction with BL1 and identified two nuclear localization signals within the N-terminal 113 residues of BR1. These nuclear localization signals were precisely located within distinct 16- and 22-peptide segments of BR1. These studies support and extend our model for squash leaf curl virus movement, showing that BR1 has a domain structure, with an N-terminal region required for nuclear targeting and a C-terminal region required for its interaction with BL1. PMID:8587985

Sanderfoot, A A; Ingham, D J; Lazarowitz, S G

1996-01-01

122

Specific interaction with the nuclear transporter importin ?2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles.  

PubMed

Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMP? proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMP?s control cellular development, we conducted a yeast two-hybrid screen for IMP?2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMP?2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMP?2-binding partner. PSPC1-IMP?2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay-based assay demonstrated direct, high-affinity PSPC1 binding to either IMP?2/IMP?1 or IMP?6/IMP?1. Coexpression of full-length PSPC1 and IMP?2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMP?2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMP?2 isoform or small interfering RNA knockdown of IMP?2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMP?2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs. PMID:25694451

Major, Andrew T; Hogarth, Cathryn A; Miyamoto, Yoichi; Sarraj, Mai A; Smith, Catherine L; Koopman, Peter; Kurihara, Yasuyuki; Jans, David A; Loveland, Kate L

2015-04-15

123

Impaired nuclear import of mammalian Dlx4 proteins as a consequence of rapid sequence divergence  

SciTech Connect

Dlx genes encode a developmentally important family of transcription factors with a variety of functions and sites of action during vertebrate embryogenesis. The murine Dlx4 gene is an enigmatic member of the family; little is known about the normal developmental function(s) of Dlx4. Here, we show that Dlx4 is expressed in the murine placenta and in a trophoblast cell line where the protein localizes to both the nucleus and cytoplasm. Despite the presence of several leucine/valine-rich motifs that match known nuclear export sequences, cytoplasmic Dlx4 is not due to CRM-1-mediated nuclear export. Rather, nuclear import of Dlx4 is compromised by specific residues that flank the nuclear localization signal. One of these residues represents a novel conserved feature of the Dlx4 protein in placental mammals, and the second represents novel variation within mouse Dlx4 isoforms. Comparison of orthologous protein sequences reveals a particularly high rate of non-synonymous change in the coding regions of mammalian Dlx4 genes. Since impaired nuclear localization is unlikely to enhance the function of a nuclear transcription factor, these data point to reduced selection pressure as the basis for the rapid divergence of the Dlx4 gene within the mammalian clade.

Coubrough, Melissa L. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Bendall, Andrew J. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada)]. E-mail: abendall@uoguelph.ca

2006-11-15

124

Subcellular distribution of nuclear import-defective isoforms of the promyelocytic leukemia protein  

PubMed Central

Background The promyelocytic leukemia (PML) protein participates in a number of cellular processes, including transcription regulation, apoptosis, differentiation, virus defense and genome maintenance. This protein is structurally organized into a tripartite motif (TRIM) at its N-terminus, a nuclear localization signal (NLS) at its central region and a C-terminus that varies between alternatively spliced isoforms. Most PML splice variants target the nucleus where they define sub-nuclear compartments termed PML nuclear bodies (PML NBs). However, PML variants that lack the NLS are also expressed, suggesting the existence of PML isoforms with cytoplasmic functions. In the present study we expressed PML isoforms with a mutated NLS in U2OS cells to identify potential cytoplasmic compartments targeted by this protein. Results Expression of NLS mutated PML isoforms in U2OS cells revealed that PML I targets early endosomes, PML II targets the inner nuclear membrane (partially due to an extra NLS at its C-terminus), and PML III, IV and V target late endosomes/lysosomes. Clustering of PML at all of these subcellular locations depended on a functional TRIM domain. Conclusions This study demonstrates the capacity of PML to form macromolecular protein assemblies at several different subcellular sites. Further, it emphasizes a role of the variable C-terminus in subcellular target selection and a general role of the N-terminal TRIM domain in promoting protein clustering. PMID:21092142

2010-01-01

125

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

126

The fate of the nuclear matrix-associated-region-binding protein SATB1 during apoptosis  

Microsoft Academic Search

Special AT-rich sequence-binding protein 1 (SATB1), predominantly expressed in thymocytes, was identified as a component of the nuclear matrix protein fraction. Programmed cell death of Jurkat T-cells was induced by various stimuli in Fas-dependent and -independent fashion. During apoptosis, but not during necrosis, SATB1 was cleaved, as rapidly as was lamin B, in a caspase-dependent way yielding a stable 70

J Gotzmann; M Meissner; C Gerner

2000-01-01

127

Identification of the Proteins of the Yeast U1 Small Nuclear Ribonucleoprotein Complex by Mass Spectrometry  

Microsoft Academic Search

Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20

Gitte Neubauer; Alexander Gottschalk; Patrizia Fabrizio; Bertrand Seraphin; Reinhard Luhrmann; Matthias Mann

1997-01-01

128

Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation  

PubMed Central

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NF?B whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NF?B action those proteins where genes have NF?B binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O’Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.

2012-01-01

129

Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.  

PubMed

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NF?B whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NF?B action those proteins where genes have NF?B binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R; Taylor, Sam; Walker, Michael; Aspinall-O'Dea, Mark; O'Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D; Poolman, Toryn; Ray, David; Whetton, Anthony D

2012-01-01

130

Early Auxin-Induced Genes Encode Short-Lived Nuclear Proteins  

Microsoft Academic Search

The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the

Steffen Abel; Paul W. Oeller; Athanasios Theologis

1994-01-01

131

The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing  

E-print Network

factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). WeRNP complex and the SF1/U2AF65/U2AF35 protein complex recognize the 59 and 39 splice sites of an intronThe Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing

Horvitz, H. Robert

132

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress  

SciTech Connect

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.

Bryantsev, A.L. [School of Molecular Biosciences, Washington State University, Pullman, WA (United States); Chechenova, M.B. [School of Molecular Biosciences, Washington State University, Pullman, WA (United States); Shelden, E.A. [School of Molecular Biosciences, Washington State University, Pullman, WA (United States) and Center for Reproductive Biology, Washington State University, Pullman, WA (United States)]. E-mail: eshelden@wsu.edu

2007-01-01

133

Galectin-3 Is a Nuclear Matrix Protein Which Binds RNA  

Microsoft Academic Search

The endogenous galectin-3 is a carbohydrate-binding protein of Mr?30,000 serving in the cytoplasm and on the cell surface as a receptor for ligands containing poly-N-acetyllactosamine sequences. In addition, galectin-3 has been demonstrated to be associated in the nucleus with ribonucleoprotein complexes and to act as a pre-mRNA splicing factor and to be involved in spliceosome assembly. However, little is known

L. Wang; H. Inohara; K. J. Pienta; A. Raz

1995-01-01

134

Cell cycle?dependent nuclear location of the matricellular protein SPARC: Association with the nuclear matrix  

Microsoft Academic Search

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that inhibits cellular adhesion and proliferation. In this study, we report the detection of SPARC in the interphase nuclei of embryonic chicken cells in vivo. Differential partitioning of SPARC was also noted in the cytoplasm of these cells during discrete stages of M-phase: cells in metaphase and anaphase

M. D. Gooden; R. B. Vernon; J. A. Bassuk; E. H. Sage

1999-01-01

135

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

Microsoft Academic Search

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

Yao E. Wang; Arnold Park; Michael Lake; Mickey Pentecost; Betsabe Torres; Tatyana E. Yun; Mike C. Wolf; Michael R. Holbrook; Alexander N. Freiberg; Benhur Lee

2010-01-01

136

Nuclear Envelope Protein Lem2 is Required for Mouse Development and Regulates MAP and AKT Kinases  

PubMed Central

The nuclear lamina, along with associated nuclear membrane proteins, is a nexus for regulating signaling in the nucleus. Numerous human diseases arise from mutations in lamina proteins, and experimental models for these disorders have revealed aberrant regulation of various signaling pathways. Previously, we reported that the inner nuclear membrane protein Lem2, which is expressed at high levels in muscle, promotes the differentiation of cultured myoblasts by attenuating ERK signaling. Here, we have analyzed mice harboring a disrupted allele for the Lem2 gene (Lemd2). No gross phenotypic defects were seen in heterozygotes, although muscle regeneration induced by cardiotoxin was delayed. By contrast, homozygous Lemd2 knockout mice died by E11.5. Although many normal morphogenetic hallmarks were observed in E10.5 knockout embryos, most tissues were substantially reduced in size. This was accompanied by activation of multiple MAP kinases (ERK1/2, JNK, p38) and AKT. Knockdown of Lem2 expression in C2C12 myoblasts also led to activation of MAP kinases and AKT. These findings indicate that Lemd2 plays an essential role in mouse embryonic development and that it is involved in regulating several signaling pathways. Since increased MAP kinase and AKT/mTORC signaling is found in other animal models for diseases linked to nuclear lamina proteins, LEMD2 should be considered to be another candidate gene for human disease. PMID:25790465

Tapia, Olga; Fong, Loren G.; Huber, Michael D.; Young, Stephen G.; Gerace, Larry

2015-01-01

137

Phylogeny of penaeoid shrimps (Decapoda: Penaeoidea) inferred from nuclear protein-coding genes  

Microsoft Academic Search

Penaeoidea is a diverse group of economically important marine shrimps. Attention to the evolutionary history of the penaeoids has been raised since studies using mitochondrial DNA markers and sperm ultrastructure contradict classification of the penaeoid families based on morphology and hence challenge the long standing taxonomy of this superfamily. In this study, DNA sequences of two nuclear protein-coding genes, phosphoenolpyruvate

K. Y. Ma; T.-Y. Chan; K. H. Chu

2009-01-01

138

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii  

PubMed Central

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes. PMID:21963440

Suvorova, Elena S.; Lehmann, Margaret M.; Kratzer, Stella; White, Michael W.

2011-01-01

139

Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport  

PubMed Central

Translationally controlled tumor protein (TCTP) lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus. When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein. PMID:22567286

Munirathinam, Gnanasekar; Ramaswamy, Kalyanasundaram

2012-01-01

140

Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes  

PubMed Central

Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1 mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment. PMID:25560244

Anavi, Sarit; Ni, Zhixu; Tirosh, Oren; Fedorova, Maria

2014-01-01

141

High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal  

Microsoft Academic Search

High-mobility group box 1 protein (HMGB1), which previously was thought to function only as a nuclear factor that enhances transcription, was recently discovered to be a crucial cytokine that mediates the response to infection, injury and inflammation. These observations have led to the emergence of a new field in immunology that is focused on understanding the mechanisms of HMGB1 release,

Michael T. Lotze; Kevin J. Tracey

2005-01-01

142

A phylogenetic analysis of Myriapoda (Arthropoda) using two nuclear protein-encoding genes  

Microsoft Academic Search

Nucleotide and inferred amino acid sequences from two nuclear protein-encoding genes, elongation factor-1? and RNA polymerase II, were obtained from 34 myriapods and 14 other arthropods to determine phylogenetic relationships among and within the myriapod classes. Phylogenetic analyses using maximum parsimony and maximum likelihood methods recovered all three represented myriapod classes (Chilopoda, Diplopoda, Symphyla) and all multiply sampled chilopod and

JEROME C. REGIER; JEFFREY W. SHULTZ

2001-01-01

143

Transcriptional Control of Receptor Activator of Nuclear Factor-B Ligand by the Protein Kinase A  

E-print Network

by a significant and location-selective rise in histone 4 acetylation. This study demon- strates relevant to the induction of RankL gene expression and its bio- logical activity continues to be of topicalTranscriptional Control of Receptor Activator of Nuclear Factor- B Ligand by the Protein Kinase

Pike, J. Wesley

144

Fanconi Anemia Proteins FANCA, FANCC, and FANCG\\/XRCC9 Interact in a Functional Nuclear Complex  

Microsoft Academic Search

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight comple- mentation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting

IRENE GARCIA-HIGUERA; YANAN KUANG; DIETER NAF; JENNIFER WASIK; ALAN D. D'ANDREA

1999-01-01

145

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

146

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

147

IQGAP1 Protein Regulates Nuclear Localization of ?-Catenin via Importin-?5 Protein in Wnt Signaling*  

PubMed Central

In the canonical Wnt signaling pathway, the translocation of ?-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of ?-catenin function via importin-?5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of ?-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-?5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and ?-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of ?-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of ?-catenin in the canonical Wnt signaling pathway. PMID:24196961

Goto, Toshiyasu; Sato, Atsushi; Adachi, Shungo; Iemura, Shun-ichiro; Natsume, Tohru; Shibuya, Hiroshi

2013-01-01

148

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

149

Nuclear PRP20 protein is required for mRNA export.  

PubMed Central

The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus. Images PMID:7679070

Amberg, D C; Fleischmann, M; Stagljar, I; Cole, C N; Aebi, M

1993-01-01

150

Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope  

PubMed Central

The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

Zattas, Dimitrios; Hochstrasser, Mark

2014-01-01

151

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

152

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

153

Protein carbon-13 spin systems by a single two-dimensional nuclear magnetic resonance experiment  

SciTech Connect

By applying a two-dimensional double-quantum carbon-13 nuclear magnetic resonance experiment to a protein uniformly enriched to 26% carbon-13, networks of directly bonded carbon atoms were identified by virtue of their one-bond spin-spin couplings and were classified by amino acid type according to their particular single- and double-quantum chemical shift patterns. Spin systems of 75 of the 98 amino acid residues in a protein, oxidized Anabaena 7120 ferredoxin (molecular weight 11,000), were identified by this approach, which represents a key step in an improved methodology for assigning protein nuclear magnetic resonance spectra. Missing spin systems corresponded primarily to residues located adjacent to the paramagnetic iron-sulfur cluster. 25 references, 2 figures.

Oh, B.H.; Westler, W.M.; Darba, P.; Markley, J.L.

1988-05-13

154

SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells  

SciTech Connect

The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M. [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)] [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Heery, David M., E-mail: david.heery@nottingham.ac.uk [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

2010-01-01

155

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

156

"Nuclear FGF receptor-1 and CREB binding protein: an integrative signaling module".  

PubMed

In this review we summarize the current understanding of a novel integrative function of Fibroblast Growth Factor Receptor-1 (FGFR1) and its partner CREB Binding Protein (CBP) acting as a nuclear regulatory complex. Nuclear FGFR1 and CBP interact with and regulate numerous genes on various chromosomes. FGFR1 dynamic oscillatory interactions with chromatin and with specific genes, underwrites gene regulation mediated by diverse developmental signals. Integrative Nuclear FGFR1 Signaling (INFS) effects the differentiation of stem cells and neural progenitor cells via the gene-controlling Feed-Forward-And-Gate mechanism. Nuclear accumulation of FGFR1 occurs in numerous cell types and disruption of INFS may play an important role in developmental disorders such as schizophrenia, and in metastatic diseases such as cancer. Enhancement of INFS may be used to coordinate the gene regulation needed to activate cell differentiation for regenerative purposes or to provide interruption of cancer stem cell proliferation. PMID:25503065

Stachowiak, Michal K; Birkaya, B; Aletta, J M; Narla, S T; Benson, C A; Decker, B; Stachowiak, E K

2015-05-01

157

Nuclear Targeting of the Tegument Protein pp65 (UL83) of Human Cytomegalovirus: an Unusual Bipartite Nuclear Localization Signal Functions with Other Portions of the Protein To Mediate Its Efficient Nuclear Transport  

Microsoft Academic Search

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion

SUSANNE SCHMOLKE; PETRA DRESCHER; GERHARD JAHN; ANDBODO PLACHTER

158

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

159

PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins  

PubMed Central

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, ?-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders. PMID:16818720

Janer, Alexandre; Martin, Elodie; Muriel, Marie-Paule; Latouche, Morwena; Fujigasaki, Hiroto; Ruberg, Merle; Brice, Alexis; Trottier, Yvon; Sittler, Annie

2006-01-01

160

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

161

Nuclear import of the yeast hexokinase 2 protein requires ?/?-importin-dependent pathway.  

PubMed

Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization and role, it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. However, the mechanism by which Hxk2 enters in the nucleus was unknown until now. Here, we report that the Hxk2 protein is an import substrate of the carriers ?-importin (Kap60 in yeast) and ?-importin (Kap95 in yeast). We also show that the Hxk2 nuclear import and the binding of Hxk2 with Kap60 are glucose-dependent and involve one lysine-rich nuclear localization sequence (NLS), located between lysine 6 and lysine 12. Moreover, Kap95 facilitates the recognition of the Hxk2 NLS1 motif by Kap60 and both importins are essential for Hxk2 nuclear import. It is also demonstrated that Hxk2 nuclear import and its binding to Kap95 and Kap60 depend on the Gsp1-GTP/GDP protein levels. Thus, our study uncovers Hxk2 as a new cargo for the ?/?-importin pathway of S. cerevisiae. PMID:22157003

Peláez, Rafael; Fernández-García, Paula; Herrero, Pilar; Moreno, Fernando

2012-01-27

162

Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila  

SciTech Connect

The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutant alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that Ya function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function might interact with itself either directly or indirectly. 27 refs., 6 figs.

Liu, Jun; Song, Kiwon; Wolfner, M.F. [Cornell Univ., Ithaca, NY (United States)

1995-12-01

163

A single herpesvirus protein can mediate vesicle formation in the nuclear envelope.  

PubMed

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission. PMID:25605719

Lorenz, Michael; Vollmer, Benjamin; Unsay, Joseph D; Klupp, Barbara G; García-Sáez, Ana J; Mettenleiter, Thomas C; Antonin, Wolfram

2015-03-13

164

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

165

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

166

Nuclear targeting determinants of the far upstream element binding protein, a c-myc transcription factor.  

PubMed

FUSE binding protein (FBP) binds in vivo and in vitro with the single-stranded far upstream element (FUSE) upstream of the c-myc gene. In addition to its transcriptional role, FBP and its closely related siblings FBP2 (KSRP) and FBP3 have been reported to bind RNA and participate in various steps of RNA processing, transport or catabolism. To perform these diverse functions, FBP must traffic to different nuclear sites. To identify determinants of nuclear localization, full-length FBP or fragments thereof were fused to green fluorescent protein. Fluorescent-FBP localized in the nucleus in three patterns, diffuse, dots and spots. Each pattern was conferred by a distinct nuclear localization signal (NLS): a classical bipartite NLS in the N-terminal and two non-canonical signals, an alpha-helix in the third KH-motif of the nucleic acid binding domain and a tyrosine-rich motif in the C-terminal transcription activation domain. Upon treatment with the transcription inhibitor actinomycin D, FBP completely re-localized into dots, but did not exit from the nucleus. This is in contrast to many general RNA-binding proteins, which shuttle from the nucleus upon treatment with actinomycin D. Furthermore, FBP co-localized with transcription sites and with the general transcription factor TFIIH, but not with the splicing factor SC-35. Taken together, these data reveal complex intranuclear trafficking of FBP and support a transcriptional role for this protein. PMID:11071946

He, L; Weber, A; Levens, D

2000-11-15

167

Interactome of the negative regulator of nuclear import BRCA1-binding protein 2  

PubMed Central

Although the negative regulator of nuclear import (NRNI) BRCA1 binding protein 2 (BRAP2) is highly expressed in testis, its role is largely unknown. Here we address this question by documenting the BRAP2 interactome from human testis, using the yeast 2-hybrid system to identify BRAP2-interacting proteins with roles in diverse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with BRAP2 in adult mouse testis with three of these, PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1), was confirmed by coimmunoprecipitation assays. BRAP2's ability to inhibit PHLPP1 and DNMT1 nuclear localisation was also confirmed by quantitative confocal microscopy. Importantly, the physiological relevance thereof was implied by the cytoplasmic localisation of PHLPP1, AKAP3 and DNMT1 in pachytene spermatocytes/round spermatids where BRAP2 is present at high levels, and nuclear localisation of PHLPP1 and DNMT1 in spermatogonia concomitant with lower levels of BRAP2. Interestingly, BRAP2 was also present in murine spermatozoa, in part colocalised with AKAP3. Together the results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold/structural role in mature spermatozoa. PMID:25820252

Fatima, Shadma; Wagstaff, Kylie M.; Loveland, Kate L.; Jans, David A.

2015-01-01

168

Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals  

Microsoft Academic Search

The interaction between phosphatidylserine and its receptor on phagocytic cells plays a critical role in the clearance of apoptotic bodies under normal physiological condition. A specific receptor for phosphatidylserine (PSR) has recently been identified by phage display and shown to mediate phosphatidylserine dependent phagocytosis. Here we show that the protein encoded by the PSR cDNA is localized in the nuclei

Ping Cui; Baoming Qin; Nan Liu; Guangjin Pan; Duanqing Pei

2004-01-01

169

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.  

PubMed Central

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase. Images PMID:6204191

Dreyfuss, G; Choi, Y D; Adam, S A

1984-01-01

170

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

171

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

172

RecQ5 protein translocation into the nucleus by a nuclear localization signal.  

PubMed

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS. PMID:23811565

Sakurai, Haruna; Tsutsui, Ayaka; Higashi, Takahiro; Azuma, Rika; Ito, Fumiaki; Kawasaki, Katsumi

2013-01-01

173

Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide.  

PubMed Central

We previously reported the purification of a complex of two proteins from human chromatin, consisting of a 47-kDa component called RCC1, which is a negative regulator of mitosis, and a 25-kDa protein. Here we show that the 25-kDa protein has a ras-related sequence. It binds guanine nucleotides, and excess Mg2+ and GDP or GTP dissociate the complex. Immunofluorescence studies and biochemical properties indicate that this polypeptide, in contrast to most members of the Ras family, is present in the nucleoplasm as a soluble monomer, in 25-fold excess over the complexed form. We designate this polypeptide Ran, for ras-related nuclear protein. Images PMID:1961752

Bischoff, F R; Ponstingl, H

1991-01-01

174

TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77*  

PubMed Central

T cell immunoglobulin and mucin domain (TIM) proteins are cell-surface signaling receptors in T cells and scavenger receptors in antigen-presenting cells and kidney tubular epithelia. Here, we demonstrated a function for TIM proteins in mediating the degradation of NUR77, a nuclear receptor implicated in the cellular response to injury. TIM proteins interacted with and mediated the lysosomal degradation of NUR77 in a phosphoinositide 3-kinase–dependent pathway. We also showed dynamic cycling of TIM-1 to and from the cell surface through clathrin-dependent constitutive endocytosis. Blocking this process or mutating the phosphatidylserine-binding pocket in TIM-1 abrogated TIM-1–mediated degradation of NUR77. In an in vitro model of kidney injury, silencing TIM-1 increased NUR77 abundance and decreased epithelial cell survival. These results show that TIM proteins may affect immune cell function and the response of the kidney to injury. PMID:23233528

Balasubramanian, Savithri; Kota, Satya Keerthi; Kuchroo, Vijay K; Humphreys, Benjamin D; Strom, Terry B

2013-01-01

175

Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin  

SciTech Connect

In a yeast artificial chromosome contig close to the nude locus on mouse chromosome 11, we identified a novel gene, nucleoredoxin, that encodes a protein with similarity to the active site of thioredoxins. Nucleoredoxin is conserved between mammalian species, and two homologous genes were found in Caenorhabditis elegans. The nucleoredoxin transcripts are expressed in all adult tissues examined, but restricted to the nervous system and the limb buds in Day 10.5-11.5 embryos. The nucleoredoxin protein is predominantly localized in the nucleus of cells transfected with the nucleoredoxin expression construct. Since the bacterially expressed protein of nucleoredoxin showed oxidoreductase activity of the insulin disulfide bonds with kinetics similar to that of thioredoxin, it may be a redox regulator of the nuclear proteins, such as transcription factors. 40 refs., 6 figs.

Kurooka, Hisanori; Kato, Keizo; Minoguchi, Shigeru [Kyoto Univ. (Japan)] [and others] [Kyoto Univ. (Japan); and others

1997-02-01

176

A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4\\/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal  

Microsoft Academic Search

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested\\/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle\\/DNA content profiles when exogenously expressed. Eight

Nadia Korfali; Vlastimil Srsen; Martin Waterfall; Dzmitry G. Batrakou; Vanja Pekovic; Christopher J. Hutchison; Eric C. Schirmer; Joanna Mary Bridger

2011-01-01

177

Characterization of a ras-related nuclear protein (Ran protein) up-regulated in shrimp antiviral immunity.  

PubMed

Diseases caused by viruses, especially white spot syndrome virus (WSSV), are the greatest challenge to worldwide shrimp aquaculture. Therefore, the innate immunity of shrimp has attracted extensive attentions these years. To date, however, no mechanism of immuno-related signal transduction pathway has been reported. In this investigation, an important signal transduction factor-Ran gene encoding ras-related nuclear protein (Ran protein) was characterized in shrimp. The shrimp Ran gene, without introns when compared with genomic DNA, was 645 bp in length. The GTP-binding assay showed that the Ran protein had GTP-binding activity. The results of RT-PCR and Western blot indicated that the transcript and protein of Ran were detected in every tissue of shrimp including hepatopancreas, haemolymph, gill, intestine, heart and muscle. In the WSSV-resistant and WSSV-infected shrimp at 4h postinfection, the Ran gene was obviously up-regulated, indicating that it played a role in shrimp immunity against virus infection. This study, therefore, might provide a clue to elucidate shrimp innate immunity. PMID:17703953

Han, Fang; Zhang, Xiaobo

2007-11-01

178

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

PubMed

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 José; Peuget, Sylvain; Kellenberger, Christine; Gravis, Gwenaëlle; Dusetti, Nelson J; Iovanna, Juan L; Rocchi, Palma; Amri, Mohamed; Carrier, Alice

2013-01-01

179

A genome-derived (gaa.ttc)24 trinucleotide block binds nuclear protein(s) specifically and forms triple helices.  

PubMed

The properties of simple trinucleotide repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. Here, we studied protein binding and structural features of a perfect (gaa.ttc)24 tract in its original genomic environment. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to the DNA fragment containing the (gaa.ttc)24 block. Competition experiments using simple (gt.ac)n repeats differing in length and flanking regions showed no cross-reactivity with the major retarded band. For the specific (gaa. ttc)n/protein complex, a binding constant of 9.3x10-9 mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa)24 strand. OsO4 and DEPC modifications followed by electrophoretic and electron microscopical analyses showed that the (gaa.ttc)24 block forms different types of intramolecular triple helices: Under superhelical stress, different H-DNA isomers are evident, whereas exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc)n tracts. PMID:9714838

Mäueler, W; Kyas, A; Keyl, H G; Epplen, J T

1998-07-30

180

Nuclear localization of DMP1 proteins suggests a role in intracellular signaling  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States) [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)] [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States)] [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D'Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)] [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)] [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

2012-08-03

181

Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex.  

PubMed

Nuclear pore complexes (NPCs) allow selective import and export while forming a barrier for untargeted proteins. Using fluorescence microscopy, we measured in vivo the permeability of the Saccharomyces cerevisiae NPC for multidomain proteins of different sizes and found that soluble proteins of 150 kDa and membrane proteins with an extralumenal domain of 90 kDa were still partly localized in the nucleus on a time scale of hours. The NPCs thus form only a weak barrier for the majority of yeast proteins, given their monomeric size. Using FG?-mutant strains, we showed that specific combinations of Nups, especially with Nup100, but not the total mass of FG-nups per pore, were important for forming the barrier. Models of the disordered phase of wild-type and mutant NPCs were generated using a one bead per amino acid molecular dynamics model. The permeability measurements correlated with the density predictions from coarse-grained molecular dynamics simulations in the center of the NPC. The combined in vivo and computational approach provides a framework for elucidating the structural and functional properties of the permeability barrier of nuclear pore complexes. PMID:25631821

Popken, Petra; Ghavami, Ali; Onck, Patrick R; Poolman, Bert; Veenhoff, Liesbeth M

2015-04-01

182

Arabidopsis thaliana atrab28: a nuclear targeted protein related to germination and toxic cation tolerance.  

PubMed

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination. PMID:12175017

Borrell, Antonio; Cutanda, M Cruz; Lumbreras, Victoria; Pujal, Judit; Goday, Adela; Culiáñez-Macià, Francisco A; Pagès, Montserrat

2002-09-01

183

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

184

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

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

2014-01-01

185

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

PubMed

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. PMID:25144938

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

2014-01-01

186

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

187

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

188

A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95  

SciTech Connect

RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.

Sugiura, Takeyuki [Discovery Research Laboratory, Tokyo R and D Center, Daiichi Pharmaceutical Co. Ltd., Daiichi-Sankyo group, 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo, 134-8630 (Japan)], E-mail: sugiura.takeyuki.uu@daiichisankyo.co.jp; Yamaguchi, Aya; Miyamoto, Kentaro [Discovery Research Laboratory, Tokyo R and D Center, Daiichi Pharmaceutical Co. Ltd., Daiichi-Sankyo group, 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo, 134-8630 (Japan)

2008-04-15

189

General organization of protein in HeLa 40S nuclear ribonucleoprotein particles  

PubMed Central

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein- RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2). PMID:3988802

1985-01-01

190

Co-localization of the amyloid precursor protein and Notch intracellular domains in nuclear transcription factories.  

PubMed

The beta-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, which may represent sites of transcription. Despite a lack of co-localization with several described nuclear compartments, we have identified a close apposition between AFT complexes and splicing speckles, Cajal bodies and PML bodies. Live imaging revealed that AFT complexes were highly mobile within nuclei and following pharmacological inhibition of transcription fused into larger assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. In support of our earlier findings, transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at 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, Zoë V; Meyer, Michelle; Kohli, Bernhard M; Bosset, Jérôme; Lahiri, Debomoy K; Nitsch, Roger M

2010-01-01

191

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

192

Different complexes are formed on the 3' end of histone mRNA with nuclear and polyribosomal proteins.  

PubMed Central

Specific protein-RNA complexes are formed by incubating a synthetic histone mRNA 3' end (a 30 nucleotide stem-loop structure) RNA with extracts of either nuclei or polyribosomes. The complex formed between the stem-loop and nuclear proteins has a lower electrophoretic mobility than the complex formed between the stem-loop and polyribosomal proteins. Binding of the synthetic 3' end by both polyribosomal and nuclear proteins is abolished when two of the conserved uridine residues in the loop are replaced with adenosines. UV crosslinking of the protein complexes to the synthetic RNA resulted in transferring radiolabel to similar sized proteins, 50 kD, in both the nuclear and polyribosomal extracts. Images PMID:1834994

Pandey, N B; Sun, J H; Marzluff, W F

1991-01-01

193

Protein profiles of bovine placenta derived from somatic cell nuclear transfer.  

PubMed

Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT. PMID:16196098

Kim, Hong Rye; Kang, Jae Ku; Yoon, Jong Taek; Seong, Hwan Hoo; Jung, Jin Kwan; Lee, Hong Mie; Sik Park, Chang; Jin, Dong Il

2005-11-01

194

Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders  

DOEpatents

The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.

Lelievre, Sophie (Berkeley, CA); Bissell, Mina (Berkeley, CA)

2001-01-01

195

Three-dimensional structures of proteins in solution by nuclear magnetic resonance spectroscopy.  

PubMed

Nuclear magnetic resonance (NMR) spectroscopy has emerged in recent years as a powerful method for the determination of three dimensional structures of small proteins in solution. Major cornerstones towards these advances were the introduction of two dimensional NMR experiments in combination with high field superconducting magnets, as well as the development of computational procedures to convert NMR derived distances into a 3D structure. This article outlines the methodology employed and illustrates its applicability based on a variety of examples. PMID:2911557

Gronenborn, A M; Clore, G M

1989-01-01

196

Transcriptional regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear factor-4  

Microsoft Academic Search

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly of triglyceride (TG)-rich apolipopro- tein B-containing liver (e.g., VLDL) and intestinal (e.g., chy- lomicron) lipoproteins. The human MTP gene promoter is reported here to associate in vivo with endogenous hepato- cyte nuclear factor-4 ? (HNF-4 ? ) and to be transactivated or transsuppressed by overexpressed or by dominant negative HNF-4 ? ,

Vered Sheena; Rachel Hertz; Janna Nousbeck; Ina Berman; Judith Magenheim; Jacob Bar-Tana

2004-01-01

197

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

198

Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness.  

PubMed

In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions. PMID:25609380

Furusawa, Takashi; Rochman, Mark; Taher, Leila; Dimitriadis, Emilios K; Nagashima, Kunio; Anderson, Stasia; Bustin, Michael

2015-01-01

199

Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness  

NASA Astrophysics Data System (ADS)

In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.

Furusawa, Takashi; Rochman, Mark; Taher, Leila; Dimitriadis, Emilios K.; Nagashima, Kunio; Anderson, Stasia; Bustin, Michael

2015-01-01

200

The Hinge of the Human Papillomavirus Type 11 E2 Protein Contains Major Determinants for Nuclear Localization and Nuclear Matrix Association  

PubMed Central

The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication. PMID:10729151

Zou, Nianxiang; Lin, Biing Yuan; Duan, Fenghai; Lee, Kyung-Yeol; Jin, Ge; Guan, Ran; Yao, Guang; Lefkowitz, Elliot J.; Broker, Thomas R.; Chow, Louise T.

2000-01-01

201

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

202

Nuclear pore proteins are involved in the biogenesis of functional tRNA.  

PubMed Central

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Images PMID:8641292

Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

1996-01-01

203

Nuclear targeting of methyl-recycling enzymes in Arabidopsis thaliana is mediated by specific protein interactions.  

PubMed

Numerous transmethylation reactions are required for normal plant growth and development. S-adenosylhomocysteine hydrolase (SAHH) and adenosine kinase (ADK) act coordinately to recycle the by-product of these reactions, S-adenosylhomocysteine (SAH) that would otherwise competitively inhibit methyltransferase (MT) activities. Here, we report on investigations to understand how the SAH produced in the nucleus is metabolized by SAHH and ADK. Localization analyses using green fluorescent fusion proteins demonstrated that both enzymes are capable of localizing to the cytoplasm and the nucleus, although no obvious nuclear localization signal was found in their sequences. Deletion analysis revealed that a 41-amino-acid segment of SAHH (Gly(150)-Lys(190)) is required for nuclear targeting of this enzyme. This segment is surface exposed, shows unique sequence conservation patterns in plant SAHHs, and possesses additional features of protein-protein interaction motifs. ADK and SAHH interact in Arabidopsis via this segment and also interact with an mRNA cap MT. We propose that the targeting of this complex is directed by the nuclear localization signal of the MT; other MTs may similarly target SAHH/ADK to other subcellular compartments to ensure uninterrupted transmethylation. PMID:21976714

Lee, Sanghyun; Doxey, Andrew C; McConkey, Brendan J; Moffatt, Barbara A

2012-01-01

204

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function.  

PubMed

Nucleocytoplasmic transport of transcription factors is essential in eukaryotes. We previously reported the presence of two functional NLSs in the homeodomain protein, aristaless-related homeobox (Arx) protein, which is a key transcriptional repressor of LMO1, SHOX2, and PAX4 during development. NLS2, that overlaps the homeodomain, is recognized directly by multiple importin ?s, but not by importin ?s. In this study, we found that the N-terminal NLS1 of Arx is targeted by multiple importin ? proteins, including importin ?3 and ?5. Both in vivo and in vitro assays demonstrated that nuclear import of Arx via NLS1 is mediated by the importin ?/? pathway. Mutagenesis analysis indicated that two basic amino acids, (84)K and (87)R, are essential to the function of NLS1, and that their mutation prevents interactions of Arx with importin ?s. Interestingly, inhibition of nuclear import of Arx via NLS1 clearly attenuates its ability of transcriptional repression, suggesting that nuclear import of Arx via NLS1 contributes to its transcriptional function. PMID:23771350

Ye, Wenduo; Lin, Wenbo; Tartakoff, Alan M; Ma, Qilin; Tao, Tao

2013-09-01

205

A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix  

PubMed Central

Telomeres are specialized heterochromatin at the ends of linear chromosomes. Telomeres are crucial for maintaining genome stability and play important roles in cellular senescence and tumor biology. Six core proteins—TRF1, TRF2, TIN2, POT1, TPP1 and Rap1 (termed the telosome or shelterin complex)—regulate telomere structure and function. One of these proteins, TIN2, regulates telomere length and structure indirectly by interacting with TRF1, TRF2 and TPP1, but no direct function has been attributed to TIN2. Here we present evidence for a TIN2 isoform (TIN2L) that differs from the originally described TIN2 isoform (TIN2S) in two ways: TIN2L contains an additional 97 amino acids, and TIN2L associates strongly with the nuclear matrix. Stringent salt and detergent conditions failed to extract TIN2L from the nuclear matrix, despite removing other telomere components, including TIN2S. In human mammary epithelial cells, each isoform showed a distinct nuclear distribution both as a function of cell cycle position and telomere length. Our results suggest a dual role for TIN2 in mediating the function of the shelterin complex and tethering telomeres to the nuclear matrix. PMID:19229133

Kaminker, Patrick G.; Kim, Sahn-Ho; Desprez, Pierre-Yves; Campisi, Judith

2009-01-01

206

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

207

The Plant TPX2 Protein Regulates Prospindle Assembly before Nuclear Envelope Breakdown[W  

PubMed Central

The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear–cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes. PMID:18941054

Vos, Jan W.; Pieuchot, Laurent; Evrard, Jean-Luc; Janski, Natacha; Bergdoll, Marc; de Ronde, Dryas; Perez, Laurent H.; Sardon, Teresa; Vernos, Isabelle; Schmit, Anne-Catherine

2008-01-01

208

MAF1, a Novel Plant Protein Interacting with Matrix Attachment Region Binding Protein MFP1, Is Located at the Nuclear Envelope  

Microsoft Academic Search

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component

Frank Gindullis; Nancy J. Peffer; Iris Meier

1999-01-01

209

FE65 regulates and interacts with the Bloom syndrome protein in dynamic nuclear spheres - potential relevance to Alzheimer's disease.  

PubMed

The intracellular domain of the amyloid precursor protein (AICD) is generated following cleavage of the precursor by the ?-secretase complex and is involved in membrane to nucleus signaling, for which the binding of AICD to the adapter protein FE65 is essential. Here we show that FE65 knockdown causes a downregulation of the protein Bloom syndrome protein (BLM) and the minichromosome maintenance (MCM) protein family and that elevated nuclear levels of FE65 result in stabilization of the BLM protein in nuclear mobile spheres. These spheres are able to grow and fuse, and potentially correspond to the nuclear domain 10. BLM plays a role in DNA replication and repair mechanisms and FE65 was also shown to play a role in DNA damage response in the cell. A set of proliferation assays in our work revealed that FE65 knockdown in HEK293T cells reduced cell replication. On the basis of these results, we hypothesize that nuclear FE65 levels (nuclear FE65/BLM containing spheres) may regulate cell cycle re-entry in neurons as a result of increased interaction of FE65 with BLM and/or an increase in MCM protein levels. Thus, FE65 interactions with BLM and MCM proteins may contribute to the neuronal cell cycle re-entry observed in brains affected by Alzheimer's disease. PMID:23572515

Schrötter, Andreas; Mastalski, Thomas; Nensa, Fabian M; Neumann, Martin; Loosse, Christina; Pfeiffer, Kathy; Magraoui, Fouzi El; Platta, Harald W; Erdmann, Ralf; Theiss, Carsten; Uszkoreit, Julian; Eisenacher, Martin; Meyer, Helmut E; Marcus, Katrin; Müller, Thorsten

2013-06-01

210

Mapping Functional Group Free Energy Patterns at Protein Occluded Sites: Nuclear Receptors and G-Protein Coupled Receptors  

PubMed Central

Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-? (PPAR?) NRs and the metabotropic glutamate (mGluR) and ?2-adreneric (?2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the ?2AR LBP were used in virtual screening to identify high efficacy agonists targeting ?2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

2015-01-01

211

Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and g-protein coupled receptors.  

PubMed

Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-? (PPAR?) NRs and the metabotropic glutamate (mGluR) and ?2-adreneric (?2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the ?2AR LBP were used in virtual screening to identify high efficacy agonists targeting ?2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

2015-03-23

212

Evolutionary conservation of nuclear and nucleolar targeting sequences in yeast ribosomal protein S6A.  

PubMed

Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5'-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs. PMID:15979583

Lipsius, Edgar; Walter, Korden; Leicher, Torsten; Phlippen, Wolfgang; Bisotti, Marc-Angelo; Kruppa, Joachim

2005-08-12

213

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

214

The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A).  

PubMed Central

The influenza virus NS1 protein inhibits the nuclear export of a spliced viral mRNA, NS2 mRNA (F. V. Alonso-Caplen, M. E. Nemeroff, Y. Qiu, and R. M. Krug, Genes Dev. 6:255-267, 1992). To identify the sequence in NS2 mRNA that is recognized by the NS1 protein, we developed a gel shift assay for the formation of specific RNA-protein complexes. With this assay, it was established that the NS1 protein binds to the poly(A) sequence at the 3' end of NS2 mRNA and of other mRNAs. In addition, the NS1 protein was shown to bind to poly(A) itself. This specificity was also observed in vivo. The NS1 protein inhibited the nuclear export of every poly(A)-containing mRNA that was tested. In contrast, the NS1 protein failed to inhibit the nuclear export of an mRNA whose 3' end was generated by cleavage without subsequent addition of poly(A). Addition of poly(A) to this mRNA enabled the NS1 protein to inhibit mRNA export. The implications of these results for the role of the NS1 protein during virus infection are discussed. Images PMID:7908060

Qiu, Y; Krug, R M

1994-01-01

215

Reverse Micelles As a Platform for Dynamic Nuclear Polarization in Solution NMR of Proteins  

PubMed Central

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ??93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules. PMID:24456213

2015-01-01

216

Evidence for two-step processing of nuclear-encoded chloroplast proteins during membrane assembly  

PubMed Central

A plastome (chloroplast genome) mutant of tobacco, lutescens-1, displays abnormal degradation of the chloroplast-encoded polypeptides which form the core complex of photosystem II (PSII). Two nuclear- encoded proteins (present in polymorphic forms), which normally function in the water oxidation process of PSII, accumulate as larger size-class polypeptides in mutant thylakoid membranes. These accumulated proteins are intermediate in size between the full-length primary protein synthesized in the cytoplasm and the proteolytically processed mature polypeptides. Trypsin treatment of unstacked mutant thylakoids and of inside-out vesicle (PSII-enriched) preparations indicated that the intermediate size forms were correctly localized on the inner surface of the thylakoid membrane, but not surface-exposed in the same way as the mature proteins. Only one of the intermediate size- class proteins could be extracted by salt washes. We interpret these data to be consistent with the idea that the two imported proteins that function in the water oxidation step of photosynthesis and are localized in the loculus (the space within the thylakoid vesicles) undergo two-step processing. The second step in proteolytic processing may be related to transport through a second membrane (the first transport step through the chloroplast envelope having been completed); this step may be arrested in the mutant due to the absence of the PSII core complex. PMID:3528170

1986-01-01

217

Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis  

PubMed Central

Ran/TC4, first identified as a well-conserved gene distantly related to H-RAS, encodes a protein which has recently been shown in yeast and mammalian systems to interact with RCC1, a protein whose function is required for the normal coupling of the completion of DNA synthesis and the initiation of mitosis. Here, we present data indicating that the nuclear localization of Ran/TC4 requires the presence of RCC1. Transient expression of a Ran/TC4 protein with mutations expected to perturb GTP hydrolysis disrupts host cell DNA synthesis. These results suggest that Ran/TC4 and RCC1 are components of a GTPase switch that monitors the progress of DNA synthesis and couples the completion of DNA synthesis to the onset of mitosis. PMID:8421051

1993-01-01

218

Extremely Long-lived Nuclear Pore Proteins in the Rat Brain+  

PubMed Central

To combat the functional decline of the proteome, cells use the process of protein turnover to replace potentially impaired polypeptides with new functional copies. Here we found that extremely long-lived proteins (ELLPs) did not turn over in post-mitotic cells of the rat central nervous system. These ELLPs were associated with chromatin and the nuclear pore complex, the central transport channels that mediate all molecular trafficking in and out of the nucleus. The longevity of these proteins would be expected to expose them to potentially harmful metabolites putting them at risk of accumulating damage over extended periods of time. Thus, it is possible that failure to maintain proper levels and functional integrity of ELLPs in non-proliferative cells might contribute to age-related deterioration in cell and tissue function. PMID:22300851

Savas, Jeffrey N.; Toyama, Brandon H.; Xu, Tao; Yates, John R.; Hetzer, Martin W.

2012-01-01

219

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

PubMed

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 and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation. PMID:15522233

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

2004-12-01

220

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence  

PubMed Central

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (v?N2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the v?N2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, v?N2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus. PMID:23761407

Ferguson, Brian J.; Benfield, Camilla T. O.; Ren, Hongwei; Lee, Vivian H.; Frazer, Gordon L.; Strnadova, Pavla; Sumner, Rebecca P.

2013-01-01

221

cGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity.  

PubMed

Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP(3)) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG Ibeta, but not PKG Ialpha, and in smooth muscle cells, IRAG and PKG Ibeta are in a complex with the IP(3) receptor at endoplasmatic reticulum membranes, where the complex regulates calcium release [Schlossmann et al., Nature, 404 (2000) 197]. We found that co-expression of IRAG and PKG Ibeta in baby hamster kidney cells prevented cGMP-induced PKG Ibeta translocation to the nucleus, and decreased cGMP/PKG Ibeta transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG Ibeta/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG Ibeta, as nuclear translocation and reporter gene activation by PKG Ialpha was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG Ibeta transcriptional activity, indicating that IRAG's effect was not explained by changes in intracellular calcium, and was not related to competition of IRAG with other PKG substrates. These results demonstrate that PKG anchoring to a specific binding protein is sufficient to dictate subcellular localization of the kinase and affect cGMP signaling in the nucleus, and may explain why nuclear translocation of PKG I does not occur in all cell types. PMID:18450420

Casteel, Darren E; Zhang, Tong; Zhuang, Shunhui; Pilz, Renate B

2008-07-01

222

cGMP-DEPENDENT PROTEIN KINASE ANCHORING BY IRAG REGULATES ITS NUCLEAR TRANSLOCATION AND TRANSCRIPTIONAL ACTIVITY  

PubMed Central

Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP3) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG I?, but not PKG I?, and in smooth muscle cells, IRAG and PKG I? are in a complex with the IP3 receptor at endoplasmatic reticulum membranes, where the complex regulates calcium release [Schlossmann et al., Nature, 404 (2000) 197]. We found that co-expression of IRAG and PKG I? in baby hamster kidney cells prevented cGMP-induced PKG I? translocation to the nucleus, and decreased cGMP/PKG I? transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG I?/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG I?, as nuclear translocation and reporter gene activation by PKG I? was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG I? transcriptional activity, indicating that IRAG’s effect was not explained by changes in intracellular calcium, and was not related to competition of IRAG with other PKG substrates. These results demonstrate that PKG anchoring to a specific binding protein is sufficient to dictate subcellular localization of the kinase and affect cGMP signaling in the nucleus, and may explain why nuclear translocation of PKG I does not occur in all cell types. PMID:18450420

Casteel, Darren E.; Zhang, Tong; Zhuang, Shunhui; Pilz, Renate B.

2008-01-01

223

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

224

The Nuclear Factor-kB Pathway Regulates Cytochrome P450 3A4 Protein Stability  

SciTech Connect

We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 is degraded by the proteasome, it seems likely that there is another protein that is susceptible to proteasomal degradation that regulates CYP3A4 expression. In this study, we evaluate whether the nuclear factor kappa B (NF-kB) pathway is involved in that process. Our model system uses an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, we found that inhibition of the proteasome with MG132 suppresses CYP3A4. Consistent with reports that proteasome inhibition suppresses the NF-kB pathway, we also observe a suppression of inhibitory kB kinase protein levels after treatment with MG132. Treatment of the HepG2 cells with NK-kB Activation Inhibitor also suppresses CYP3A4 proteins levels. In contrast, inhibition of either the proteasome or NF-kB pathways increases CYP3A4 mRNA levels. When the HepG2 cells are treated with cycloheximide, a general inhibitor of translation, the loss of CYP3A4 protein is accelerated by co-treatment with an NF-kB Activation Inhibitor. These results indicate that NF-kB activity regulates CYP3A4 protein stability and suggest that the NF-kB pathway is responsible for the decrease in CYP3A4 protein levels that results from the inhibition of proteasomal activity.

Zangar, Richard C.; Bollinger, Nikki; Verma, Seema; Karin, Norm J.; Lu, Yi

2008-06-01

225

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium.  

PubMed

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases. PMID:2426061

Oud, P S; Reubsaet-Veldhuizen, J A; Beck, H L; Pahlplatz, M M; Hesselmans, G H; Hermkens, H G; Tas, J; James, J; Vooijs, G P

1986-07-01

226

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

E-print Network

expression (Kendrick and Kronenberg, 1994). However, except for phyB, the cellular localization of most phytochrome species remains unclear. phyB has been shown to be a nuclear protein and the nuclear localization of phyB was dependent on light (Sakamoto and Nagatani, 1996). Dark treatment of light-grown plants before

Lin, Chentao

227

Nuclear Matrix Proteins Associated with DNA in Situ in Hormone-dependent and Hormone-independent Human Breast Cancer Cell Lines1  

Microsoft Academic Search

The nuclear matrix is a dynamic RNA-protein complex that organizes chromatin and regulates nuclear DNA metabolism. Nuclear matrix pro- teins informative in the diagnosis of cancer have been identified. Here, the nuclear matrix breast cancer proteins (NMBCs) cross-linked to nuclear DNA in situ with cisplatin in human breast cancer cell lines were analyzed by two-dimensional gel electrophoresis. We identified NMBCs

Virginia A. Spencer; Shanti K. Samuel; James R. Davie

2000-01-01

228

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation.  

PubMed

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS. PMID:16014412

Yang, Shao H; Bergo, Martin O; Toth, Julia I; Qiao, Xin; Hu, Yan; Sandoval, Salemiz; Meta, Margarita; Bendale, Pravin; Gelb, Michael H; Young, Stephen G; Fong, Loren G

2005-07-19

229

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson–Gilford progeria syndrome mutation  

PubMed Central

Hutchinson–Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a “CAAX protein” that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by ?2 statistic). These studies suggest a possible treatment strategy for HGPS. PMID:16014412

Yang, Shao H.; Bergo, Martin O.; Toth, Julia I.; Qiao, Xin; Hu, Yan; Sandoval, Salemiz; Meta, Margarita; Bendale, Pravin; Gelb, Michael H.; Young, Stephen G.; Fong, Loren G.

2005-01-01

230

Nuclear localization of Klotho in brain: an anti-aging protein  

PubMed Central

Klotho is a putative age-suppressing gene whose over-expression in mice results in extension of life span. The klotho gene encodes a single-pass transmembrane protein whose extracellular domain is shed and released into blood, urine, and cerebrospinal fluid, potentially functioning as a humoral factor. The extracellular domain of Klotho has an activity that increases the expression of anti-oxidant enzymes and confers resistance to oxidative stress in cultured cells and in whole animals. The transmembrane form of the Klotho protein directly binds to multiple fibroblast growth factor receptors and modifies their ligand affinity and specificity. The purpose of the present study was to determine the precise cellular localization of Klotho in the mouse brain. Using light microscopic immunohistochemical methods, we found the highest levels of Klotho immunoreactivity in two brain regions: the choroid plexus, and cerebellar Purkinje cells. In the choroid plexus cells, Klotho was found not only on the plasma membrane but also in large amounts near the nuclear membrane. Likewise, in the Purkinje cell Klotho was found throughout the cell including dendrites, axon and soma with large amounts near the nuclear membrane. Using immunoelectron microscopy, we found Klotho in the cell membrane, but the highest concentration was localized in the peripheral portion of the nucleus and the nucleolus in both cell types. This new finding suggests that in addition to Klotho being secreted from cells in brain, it also has a nuclear function. PMID:22245317

German, Dwight C.; Khobahy, Ida; Pastor, Johanne; Kuro-o, Makoto; Liu, Xinran

2011-01-01

231

The C terminus of the nuclear protein NuMA: Phylogenetic distribution and structure  

PubMed Central

The C terminus of the nuclear protein NuMA, NuMA-CT, has a well-known function in mitosis via its proximal segment, but it seems also involved in the control of differentiation. To further investigate the structure and function of NuMA, we exploited established computational techniques and tools to collate and characterize proteins with regions similar to the distal portion of NuMA-CT (NuMA-CTDP). The phylogenetic distribution of NuMA-CTDP was examined by PSI-BLAST- and TBLASTN-based analysis of genome and protein sequence databases. Proteins and open reading frames with a NuMA-CTDP-like region were found in a diverse set of vertebrate species including mammals, birds, amphibia, and early teleost fish. The potential structure of NuMA-CTDP was investigated by searching a database of protein sequences of known three-dimensional structure with a hidden Markov model (HMM) estimated using representative (human, frog, chicken, and pufferfish) sequences. The two highest scoring sequences that aligned to the HMM were the extracellular domains of ?3-integrin and Her2, suggesting that NuMA-CTDP may have a primarily ? fold structure. These data indicate that NuMA-CTDP may represent an important functional sequence conserved in vertebrates, where it may act as a receptor to coordinate cellular events. PMID:15388855

Abad, Patricia C.; Mian, I. Saira; Plachot, Cedric; Nelpurackal, Aniysha; Bator-Kelly, Carol; Lelièvre, Sophie A.

2004-01-01

232

The C terminus of the nuclear protein NuMA: phylogenetic distribution and structure.  

PubMed

The C terminus of the nuclear protein NuMA, NuMA-CT, has a well-known function in mitosis via its proximal segment, but it seems also involved in the control of differentiation. To further investigate the structure and function of NuMA, we exploited established computational techniques and tools to collate and characterize proteins with regions similar to the distal portion of NuMA-CT (NuMA-CTDP). The phylogenetic distribution of NuMA-CTDP was examined by PSI-BLAST- and TBLASTN-based analysis of genome and protein sequence databases. Proteins and open reading frames with a NuMA-CTDP-like region were found in a diverse set of vertebrate species including mammals, birds, amphibia, and early teleost fish. The potential structure of NuMA-CTDP was investigated by searching a database of protein sequences of known three-dimensional structure with a hidden Markov model (HMM) estimated using representative (human, frog, chicken, and pufferfish) sequences. The two highest scoring sequences that aligned to the HMM were the extracellular domains of beta3-integrin and Her2, suggesting that NuMA-CTDP may have a primarily beta fold structure. These data indicate that NuMA-CTDP may represent an important functional sequence conserved in vertebrates, where it may act as a receptor to coordinate cellular events. PMID:15388855

Abad, Patricia C; Mian, I Saira; Plachot, Cedric; Nelpurackal, Aniysha; Bator-Kelly, Carol; Lelièvre, Sophie A

2004-10-01

233

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

234

DETECTION OF EXTRA-NUCLEAR HIGH MOBILITY GROUP BOX-1 PROTEIN IN A CANINE MODEL OF MYOCARDIAL INFARCTION  

Technology Transfer Automated Retrieval System (TEKTRAN)

The high mobility group box-1 protein (HMGB-1) is a well-characterized nuclear protein recently shown to be involved in endotoxin-induced inflammation and injury. Studies have linked HMGB-1 release to the production of pro-inflammatory cytokines; however, a role for HMGB-1 in other disorders involvi...

235

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

236

The shelterin protein POT-1 anchors Caenorhabditis elegans telomeres through SUN-1 at the nuclear periphery  

PubMed Central

Telomeres are specialized protein–DNA structures that protect chromosome ends. In budding yeast, telomeres form clusters at the nuclear periphery. By imaging telomeres in embryos of the metazoan Caenorhabditis elegans, we found that telomeres clustered only in strains that had activated an alternative telomere maintenance pathway (ALT). Moreover, as in yeast, the unclustered telomeres in wild-type embryos were located near the nuclear envelope (NE). This bias for perinuclear localization increased during embryogenesis and persisted in differentiated cells. Telomere position in early embryos required the NE protein SUN-1, the single-strand binding protein POT-1, and the small ubiquitin-like modifier (SUMO) ligase GEI-17. However, in postmitotic larval cells, none of these factors individually were required for telomere anchoring, which suggests that additional mechanisms anchor in late development. Importantly, targeted POT-1 was sufficient to anchor chromatin to the NE in a SUN-1–dependent manner, arguing that its effect at telomeres is direct. This high-resolution description of telomere position within C. elegans extends our understanding of telomere organization in eukaryotes. PMID:24297748

Ferreira, Helder C.; Towbin, Benjamin D.; Jegou, Thibaud

2013-01-01

237

Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35  

SciTech Connect

The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.

Salsman, Jayme; Wang Xueqi; Frappier, Lori, E-mail: lori.frappier@utoronto.ca

2011-06-05

238

Purification and properties of a 90-kDa nuclear actin-binding protein.  

PubMed

A 90 kDa actin-binding nuclear protein (ABNP) with a pI of 5.2 has been purified from the 0.7 M NaCl extracted residue fraction of chromatin prepared from Novikoff hepatoma cell nuclei. This residue fraction was previously shown to contain nuclear actin. Although twice the size, similar in pI, and similar in amino acid composition to actin, the tryptic peptide map for ABNP is distinct and contains the appropriate number of tyrosine-containing tryptic peptides for a protein of 90,000 molecular weight. A comparison of the amino acid composition of ABNP with those reported in the literature for gelsolin and villin, using a calculation of S delta Q as an indication of relatedness, results in values of 30 and 27, respectively. Actin-binding activity, however, was demonstrated for both crude and gel purified ABNP using a gel-overlay technique that employs 125I-G-actin to detect specific actin-binding proteins. PMID:3952079

Yeoman, L C; Bremer, J W

1986-04-01

239

Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins  

PubMed Central

The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest. PMID:24523288

Guo, Yuxuan; Kim, Youngjo; Shimi, Takeshi; Goldman, Robert D.; Zheng, Yixian

2014-01-01

240

Nuclear protein phosphatase-1: an epigenetic regulator of fear memory and amygdala long-term potentiation.  

PubMed

Complex brain diseases and neurological disorders in human generally result from the disturbance of multiple genes and signaling pathways. These disturbances may derive from mutations, deletions, translocations or rearrangements of specific gene(s). However, over the past years, it has become clear that such disturbances may also derive from alterations in the epigenome affecting several genes simultaneously. Our work recently demonstrated that epigenetic mechanisms in the adult brain are in part regulated by protein phosphatase 1 (PP1), a protein Ser/Thr phosphatase that negatively regulates hippocampus-dependent long-term memory (LTM) and synaptic plasticity. PP1 is abundant in brain structures involved in emotional processing like the amygdala, it may therefore be involved in the regulation of fear memory, a form of memory related to post-traumatic stress disorder (PTSD) in human. Here, we demonstrate that PP1 is a molecular suppressor of fear memory and synaptic plasticity in the amygdala that can control chromatin remodeling in neurons. We show that the selective inhibition of the nuclear pool of PP1 in amygdala neurons significantly alters posttranslational modifications (PTMs) of histones and the expression of several memory-associated genes. These alterations correlate with enhanced fear memory, and with an increase in long-term potentiation (LTP) that is transcription-dependent. Our results underscore the importance of nuclear PP1 in the amygdala as an epigenetic regulator of emotional memory, and the relevance of protein phosphatases as potential targets for therapeutic treatment of brain disorders like PTSD. PMID:21093547

Koshibu, K; Gräff, J; Mansuy, I M

2011-01-26

241

Characteristics of Nucleocytoplasmic Transport of H1N1 Influenza A Virus Nuclear Export Protein  

PubMed Central

ABSTRACT The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference. IMPORTANCE Because the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains. PMID:24741105

Gao, Shengyan; Wang, Shanshan; Cao, Shuai; Sun, Lei; Li, Jing; Bi, Yuhai; Gao, George F.

2014-01-01

242

Nuclear pore complex proteins mark the implantation window in human endometrium  

PubMed Central

Summary Nucleolar channel systems (NCSs) are membranous organelles appearing transiently in the epithelial cell nuclei of postovulatory human endometrium. Their characterization and use as markers for a healthy receptive endometrium have been limited because they are only identifiable by electron microscopy. Here we describe the light microscopic detection of NCSs using immunofluorescence. Specifically, the monoclonal nuclear pore complex antibody 414 shows that NCSs are present in about half of all human endometrial epithelial cells but not in any other cell type, tissue or species. Most nuclei contain only a single NCS of uniform 1 ?m diameter indicating a tightly controlled organelle. The composition of NCSs is as unique as their structure; they contain only a subset each of the proteins of nuclear pore complexes, inner nuclear membrane, nuclear lamina and endoplasmic reticulum. Validation of our robust NCS detection method on 95 endometrial biopsies defines a 6-day window, days 19-24 (±1) of an idealized 28 day cycle, wherein NCSs occur. Therefore, NCSs precede and overlap with the implantation window and serve as potential markers of uterine receptivity. The immunodetection assay, combined with the hitherto underappreciated prevalence of NCSs, now enables simple screening and further molecular and functional dissection. PMID:18505792

Guffanti, Elisa; Kittur, Nupur; Brodt, Z. Nilly; Polotsky, Alex J.; Kuokkanen, Satu M.; Heller, Debra S.; Young, Steven L.; Santoro, Nanette; Meier, U. Thomas

2009-01-01

243

The Germ Cell Nuclear Proteins hnRNP GT and RBMY Activate a Testis-Specific Exon  

Microsoft Academic Search

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals.

Yilei Liu; Cyril F. Bourgeois; Shaochen Pang; Marek Kudla; Natacha Dreumont; Liliane Kister; Yong-Hua Sun; James Stevenin; David J. Elliott

2009-01-01

244

Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins  

Microsoft Academic Search

The PML protein is associated to nuclear bodies (NBs) whose functions are as yet unknown. PML and two other NBs-associated proteins, Sp100 And ISG20 are directly induced by interferons (IFN). PML and Sp100 proteins are covalently linked to SUMO-1, and ubiquitin-like peptide. PML NBs are disorganized in acute promyelocytic leukemia and during several DNA virus infections. In particular, the HSV-1

Mounira K Chelbi-Alix; Hugues de Thé

1999-01-01

245

Genome-Wide Screen of Three Herpesviruses for Protein Subcellular Localization and Alteration of PML Nuclear Bodies  

Microsoft Academic Search

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein–Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve

Jayme Salsman; Nicole Zimmerman; Tricia Chen; Megan Domagala; Lori Frappier

2008-01-01

246

Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.  

PubMed

In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation. PMID:25274774

Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Oma, Yoko; Hattori, Nobutaka; Ishiura, Shoichi; Nukina, Nobuyuki

2015-02-01

247

Collapsin response mediator protein 3 deacetylates histone H4 to mediate nuclear condensation and neuronal death  

PubMed Central

CRMP proteins play critical regulatory roles during semaphorin-mediated neurite outgrowth, neuronal differentiation and death. Albeit having a high degree of structure and sequence resemblance to that of liver dihydropyrimidinase, purified rodent brain CRMPs do not hydrolyze dihydropyrimidinase substrates. Here we found that mouse CRMP3 has robust histone H4 deacetylase activity. During excitotoxicity-induced mouse neuronal death, calpain-cleaved, N-terminally truncated CRMP3 undergoes nuclear translocation to cause nuclear condensation through deacetylation of histone H4. CRMP3-mediated deacetylation of H4 leads to de-repression of the E2F1 gene transcription and E2F1-dependent neuronal death. These studies revealed a novel mechanism of CRMP3 in neuronal death. Together with previous well established bodies of literature that inhibition of histone deacetylase activity provides neuroprotection, we envisage that inhibition of CRMP3 may represent a novel therapeutic approach towards excitotoxicity-induced neuronal death. PMID:23443259

Hou, Sheng T.; Jiang, Susan X.; Aylsworth, Amy; Cooke, Matthew; Zhou, Lei

2013-01-01

248

A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1  

PubMed Central

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways. PMID:8794858

1996-01-01

249

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.  

PubMed

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2? phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2? after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. PMID:25329362

Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

2015-04-01

250

Expression and nuclear localization of the TATA-box-binding protein during baculovirus infection.  

PubMed

The TATA-box-binding protein (TBP) plays a key role in initiating eukaryotic transcription and is used by many viruses for viral transcription. We previously reported increased TBP levels during infection with the baculovirus Autographa californica multicapsid nuclear polyhedrovirus (AcMNPV). The TBP antiserum used in that study, however, cross-reacted with a baculoviral protein. Here, we reported that increased amounts of nuclear TBP were detected upon infection of Spodoptera frugiperda and TN-368 cells with a TBP-specific antiserum. TBP levels increased until 72 h post-infection (p.i.), whilst tbp transcripts decreased by 16 h p.i., which suggested a virus-induced influence on the TBP protein levels. To address a potential modification of the TBP degradation pathway during infection, we investigated the possible role of viral ubiquitin. Infection studies with AcMNPV recombinants carrying a mutated viral ubiquitin gene revealed that the TBP increase during infection was not altered. In addition, pulse-chase experiments indicated a high TBP half-life of ~60 h in uninfected cells, suggesting that a virus-induced increase of TBP stability was unlikely. This increase in TBP correlated with a redistribution to nuclear domains resembling sites of viral DNA synthesis. Furthermore, we observed colocalization of TBP with host RNA polymerase (RNAP) II, but only until 8 h p.i., whilst TBP, but not RNAPII, was present in the enlarged replication domains late during infection. Thus, we suggested that AcMNPV adapted a mechanism to accumulate the highly stable cellular TBP at sites of viral DNA replication and transcription. PMID:24676420

Mainz, Daniela; Quadt, Ilja; Stranzenbach, Andrea K; Voss, Daniel; Guarino, Linda A; Knebel-Mörsdorf, Dagmar

2014-06-01

251

Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene.  

PubMed Central

To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 'forkhead' family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8216221

Bingle, C D; Gitlin, J D

1993-01-01

252

Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX  

SciTech Connect

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Iwahara, Naotoshi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)] [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Hisahara, Shin; Hayashi, Takashi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan) [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University, Sapporo 060-8556 (Japan); Horio, Yoshiyuki, E-mail: horio@sapmed.ac.jp [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)] [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)

2009-09-04

253

Nuclear Matrix Protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB  

E-print Network

(Senderek et al, 2009) and amyotrophic lateral sclerosis (ALS) (Johnson et al, 2014). Matrin3 is located diffusely throughout the nucleoplasm and is concentrated in the nuclear scaffold (Zeitz et al, 2009), and its DNA- and RNA- binding domains suggest... ). Matrin has also been shown to be involved in the early stages of the DNA double-strand break response (Salton et al, 2010). A number of functional roles A B D C Figure 1. PTB RRM2 interacts with multiple RNA-binding proteins. A Schematic representation...

Coelho, Miguel B.; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher W.J.

2015-01-19

254

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure  

Microsoft Academic Search

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP393-408 (htNASP393–408), were investigated for their antifertility effect. Active immunization with rmtNASP or

Min Wang; Jian-Li Shi; Guo-Yan Cheng; Yan-Qing Hu; Chen Xu

2009-01-01

255

Iterative Development of an Application to Support Nuclear Magnetic Resonance Data Analysis of Proteins  

PubMed Central

The CONNecticut Joint University Research (CONNJUR) team is a group of biochemical and software engineering researchers at multiple institutions. The vision of the team is to develop a comprehensive application that integrates a variety of existing analysis tools with workflow and data management to support the process of protein structure determination using Nuclear Magnetic Resonance (NMR). The use of multiple disparate tools and lack of data management, currently the norm in NMR data processing, provides strong motivation for such an integrated environment. This manuscript briefly describes the domain of NMR as used for protein structure determination and explains the formation of the CONNJUR team and its operation in developing the CONNJUR application. The manuscript also describes the evolution of the CONNJUR application through four prototypes and describes the challenges faced while developing the CONNJUR application and how those challenges were met. PMID:22214925

Ellis, Heidi J. C.; Nowling, Ronald J.; Vyas, Jay; Martyn, Timothy O.; Gryk, Michael R.

2011-01-01

256

Cross-linking and immunoprecipitation of nuclear RNA-binding proteins.  

PubMed

The systematic identification of in vivo targets of nuclear RNA-binding proteins (RBPs) is crucial to elucidate the physiological functions of each RBP. However, it has been difficult to distinguish real targets from nonspecifically bound RNAs and to determine the exact binding sites of each RBP by using a conventional RNA-immunoprecipitation (RIP) method. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) is a recently developed method that relies on RNA-protein cross-linking to reduce the contamination of nonspecifically bound RNAs. Furthermore, in combination with high-throughput sequencing followed by bioinformatic analysis, the exact RBP-binding sites can be identified at a single nucleotide resolution. Here, we describe in detail a PAR-CLIP protocol to prepare cDNA libraries for high-throughput sequencing from RNA fragments that are bound to RBPs not only in the nucleus but also in the cytoplasm. PMID:25555586

Li, Quan; Uemura, Yuri; Kawahara, Yukio

2015-01-01

257

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

258

Nuclear magnetic resonance analysis of the acetylation pattern of the neuronal Tau protein.  

PubMed

Lysine acetylation of the neuronal Tau protein was described as a novel mechanism of posttranslational regulation of Tau functions with important outcomes in microtubule binding and aggregation processes related to Alzheimer's disease. Here, we unravel at a per-residue resolution the acetylation pattern of full-length Tau by the Creb-binding protein (CBP) acetyltransferase using high-resolution nuclear magnetic resonance spectroscopy. Our study gives a quantitative overview of CBP-mediated acetylation and examines the catalytic proficiency because the nonenzymatic reaction with acetyl-coenzyme A occurs in vitro. Furthermore, we have investigated with this characterized acetylated Tau the effect of acetylation on Tau fibrillization in a heparin-induced aggregation assay and on heparin binding. PMID:24708343

Kamah, Amina; Huvent, Isabelle; Cantrelle, François-Xavier; Qi, Haoling; Lippens, Guy; Landrieu, Isabelle; Smet-Nocca, Caroline

2014-05-13

259

Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages  

PubMed Central

A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of ?29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of ?29 TP led us to identify a bona fide NLS within residues 1–37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of ?29 TP attached to the 5? DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes. PMID:23091024

Redrejo-Rodríguez, Modesto; Muñoz-Espín, Daniel; Holguera, Isabel; Mencía, Mario; Salas, Margarita

2012-01-01

260

Toward the quantum chemical calculation of nuclear magnetic resonance chemical shifts of proteins.  

PubMed

Despite the many protein structures solved successfully by nuclear magnetic resonance (NMR) spectroscopy, quality control of NMR structures is still by far not as well established and standardized as in crystallography. Therefore, there is still the need for new, independent, and unbiased evaluation tools to identify problematic parts and in the best case also to give guidelines that how to fix them. We present here, quantum chemical calculations of NMR chemical shifts for many proteins based on our fragment-based quantum chemical method: the adjustable density matrix assembler (ADMA). These results show that (13)C chemical shifts of reasonable accuracy can be obtained that can already provide a powerful measure for the structure validation. (1)H and even more (15)N chemical shifts deviate more strongly from experiment due to the insufficient treatment of solvent effects and conformational averaging. PMID:21557322

Frank, Andrea; Onila, Ionut; Möller, Heiko M; Exner, Thomas E

2011-07-01

261

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

262

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. PMID:25396176

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

263

A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein.  

PubMed

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986

Li, Zhuo; Huang, Richard Y-C; Yopp, Daniel C; Hileman, Travis H; Santangelo, Thomas J; Hurwitz, Jerard; Hudgens, Jeffrey W; Kelman, Zvi

2014-05-01

264

Smad nuclear interacting protein, SNIP1, mediates the ecdysteroid signal transduction in red crayfish Procambarus clarkii.  

PubMed

A smad nuclear interacting protein 1 (SNIP1) homolog was identified in the crayfish Procambarus clarkii and this gene encoded a polypeptide of 288 amino acids. Phylogenic analysis showed that P. clarkii SNIP1 was highly homologous to that of invertebrates and shared a highly conserved FHA domain of SNIP proteins at the C-terminus. Quantitative real-time PCR (qRT-PCR) analysis showed that Pc-SNIP1 expression was higher in heart than that in other examined tissues. In addition, prokaryotic expression and purification of the recombinant SNIP1 protein were performed. SDS-PAGE and western blot analysis demonstrated that a 35?KDa recombinant protein was successfully expressed in Escherichia coli cells. The expression of Pc-SNIP1 was significantly up-regulated in hepatopancreas after 20-hydroxyecdysone induction. Knockdown of Pc-SNIP1 gene by small interfering RNA (siRNA) transfection had significant influence on the expression of some ecdysteroid-responsive genes in hepatopancreas, which were confirmed by qRT-PCR and western blot. All together, these results suggest that SNIP1 is involved in the physiological process regulated by ecdysteroid in P. clarkii. J. Exp. Zool. 323A: 128-136, 2015. © 2015 Wiley Periodicals, Inc. PMID:25678477

Wang, Daojun; Peng, Tao; Yu, Yingying; Liu, Chao-Liang; Zhu, Bao-Jian

2015-02-01

265

Identification and characterization of a protein kinase gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.  

PubMed Central

The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding vPK has been cloned and sequenced. LdMNPV vPK shows a 24% amino acid identity to the catalytic domains of the eucaryotic protein kinases nPKC from rabbits, HSPKCE from humans, APLPKCB from Aplysia californica, and dPKC98F from Drosophila melanogaster, and homology to several other protein kinases from yeasts, mice, and bovines. The homology suggests that vPK is a serine/threonine protein kinase as defined by Hanks et al. (S.K. Hanks, A.M. Quinn, and T. Hunter, Science 241:42-52, 1988). Temporal expression studies indicate that vPK is expressed throughout the infection cycle beginning at 4 h postinfection, first as a delayed-early gene and subsequently as a late gene. Sequence analysis and primer extension reactions confirm the presence of distinct early and late transcription initiation regions. Expression of vPK with a rabbit reticulocyte system generated a 31-kDa protein, which is in close agreement with the predicted size of 32 kDa from the amino acid sequence. Phosphorylation activity of in vitro-expressed vPK was demonstrated by using calf thymus histones. Images PMID:8107234

Bischoff, D S; Slavicek, J M

1994-01-01

266

Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.  

PubMed

Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

2014-06-01

267

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

268

A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein  

PubMed Central

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986

Li, Zhuo; Huang, Richard Y.-C.; Yopp, Daniel C.; Hileman, Travis H.; Santangelo, Thomas J.; Hurwitz, Jerard; Hudgens, Jeffrey W.; Kelman, Zvi

2014-01-01

269

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity  

PubMed Central

The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among three Drosophila Piwi proteins—called Piwi, Aubergine, and Argonaute 3—Piwi is the only member of this subfamily that is localized in the nucleus and expressed in both germ-line and somatic cells in the gonad, where it is responsible for piRNA biogenesis and regulatory functions essential for fertility. In this study, we demonstrate beyond doubt that the slicer activity of Piwi is not required for any known functions in vivo. We show that, in transgenic flies with the DDX catalytic triad of PIWI mutated, neither primary nor secondary piRNA biogenesis is detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi is independent of its in vitro endonuclease activity. Instead, it is consistent with the alternative mode of Piwi function as a molecule involved in the piRNA-directed guidance of epigenetic factors to chromatin. PMID:23297219

Darricarrère, Nicole; Liu, Na; Watanabe, Toshiaki; Lin, Haifan

2013-01-01

270

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins  

PubMed Central

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs. PMID:23115280

Huang, Shengping; Chen, Jingjing; Chen, Quanjiao; Wang, Huadong; Yao, Yanfeng; Chen, Jianjun

2013-01-01

271

Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation  

SciTech Connect

Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

Cambier, Linda [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)] [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France); Pomies, Pascal, E-mail: pascal.pomies@crbm.cnrs.fr [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)] [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)

2011-06-17

272

Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes.  

PubMed Central

The interaction between cellular factors and polyoma virus (Py) DNA was investigated by using a gel retention assay. Nuclear extracts from various cell lines (NIH 3T3, NIH 3T6, LTK-, F9) contained proteins that formed specific and distinct complexes with Py B enhancer fragments of either wild-type or F9-1 mutant origin. The presence of an excess amount of other well-characterized DNA sequences, including the Py A enhancer, the murine sarcoma virus enhancer, and the simian virus 40 enhancer-promoter region, did not interfere with this protein-DNA interaction. However, a fragment previously defined as containing the lymphotropic papovavirus enhancer shares the binding of some common factor. This observation, in combination with the results of retention gel assays at different Mg2+ concentrations, indicates the interaction of several nuclear factors and Py DNA. The assay systems that were used allowed a distinction between some factors on the basis of their different biochemical and sequence requirements. The contact sites of these complexes were mapped to the B enhancer region of Py with Bal 31-derived mutant restriction fragments and ExoIII nuclease and are compatible with the functional domains determined in vivo. Images PMID:3023889

Böhnlein, E; Gruss, P

1986-01-01

273

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

PubMed Central

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.

2014-01-01

274

Human Macrophage Inflammatory Protein 3?: Protein and Peptide Nuclear Magnetic Resonance Solution Structures, Dimerization, Dynamics, and Anti-Infective Properties? †  

PubMed Central

Human macrophage inflammatory protein 3? (MIP-3?), also known as CCL20, is a 70-amino-acid chemokine which exclusively binds to chemokine receptor 6. In addition, the protein also has direct antimicrobial, antifungal, and antiviral activities. The solution structure of MIP-3? was solved by the use of two-dimensional homonuclear proton nuclear magnetic resonance (NMR). The structure reveals the characteristic chemokine fold, with three antiparallel ? strands followed by a C-terminal ? helix. In contrast to the crystal structures of MIP-3?, the solution structure was found to be monomeric. Another difference between the NMR and crystal structures lies in the angle of the ? helix with respect to the ? strands, which measure 69 and ?56.5° in the two structures, respectively. NMR diffusion and pH titration studies revealed a distinct tendency for MIP-3? to form dimers at neutral pH and monomers at lower pH, dependent on the protonation state of His40. Molecular dynamics simulations of both the monomeric and the dimeric forms of MIP-3? supported the notion that the chemokine undergoes a change in helix angle upon dimerization and also highlighted the important hydrophobic and hydrogen bonding contacts made by His40 in the dimer interface. Moreover, a constrained N terminus and a smaller binding groove were observed in dimeric MIP-3? simulations, which could explain why monomeric MIP-3? may be more adept at receptor binding and activation. The solution structure of a synthetic peptide consisting of the last 20 residues of MIP-3? displayed a highly amphipathic ? helix, reminiscent of various antimicrobial peptides. Antimicrobial assays with this peptide revealed strong and moderate bactericidal activities against Escherichia coli and Staphylococcus aureus, respectively. This confirms that the C-terminal ?-helical region of MIP-3? plays a significant part in its broad anti-infective activity. PMID:18086840

Chan, David I.; Hunter, Howard N.; Tack, Brian F.; Vogel, Hans J.

2008-01-01

275

Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.  

PubMed Central

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals. PMID:8524271

Feldherr, C; Akin, D

1995-01-01

276

The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains.  

PubMed

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein. PMID:8970944

Yu, S F; Edelmann, K; Strong, R K; Moebes, A; Rethwilm, A; Linial, M L

1996-12-01

277

Identification of a Nuclear Export Signal Sequence for Bovine Papillomavirus E1 Protein  

PubMed Central

Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over or under expression of the Crm1, the major cellular exportin, and export was strongly reduced by the Crm1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a Crm1-dependent process. Consistent with the in vivo functional results, E1 bound Crm1 in an in vitro pulldown assays. In addition, sumoylated E1 bound Crm1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wildtype E1, yet was defective for viral origin replication, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state. PMID:18201744

Rosas-Acosta, Germán; Wilson, Van G.

2008-01-01

278

Nuclear TAR DNA-binding protein 43: A new target for amyotrophic lateral sclerosis treatment  

PubMed Central

Abnormal TAR DNA-binding protein 43 (TDP-43) inclusion bodies can be detected in the degenerative neurons of amyotrophic lateral sclerosis. In this study, we induced chronic oxidative stress injury by applying malonate to cultured mouse cortical motor neurons. In the later stages of the malonate insult, TDP-43 expression reduced in the nuclei and transferred to the cytoplasm. This was accompanied by neuronal death, mimicking the pathological changes in TDP-43 that are seen in patients with amyotrophic lateral sclerosis. Interestingly, in the early stages of the response to malonate treatment, nuclear TDP-43 expression increased, and neurons remained relatively intact, without inclusion bodies or fragmentation. Therefore, we hypothesized that the increase of nuclear TDP-43 expression might be a pro-survival factor against oxidative stress injury. This hypothesis was confirmed by an in vitro transgenic experiment, in which overexpression of wild type mouse TDP-43 in cultured cortical motor neurons significantly reduced malonate-induced neuronal death. Our findings suggest that the loss of function of TDP-43 is an important cause of neuronal degeneration, and upregulation of nuclear TDP-43 expression might be neuroprotective in amyotrophic lateral sclerosis. PMID:25206650

Zheng, Mei; Shi, Yujie; Fan, Dongsheng

2013-01-01

279

A monoclonal antibody against nuclear lamina proteins reveals cell type-specificity in Xenopus laevis.  

PubMed

Immunofluorescence microscopy shows that the monoclonal murine antibody PKB8 stains the nuclear lamina of various somatic cells from vertebrates as diverse as mammals, birds and amphibia. It also decorates the nuclear periphery of oocytes from rat and chicken but does not react with spermatocytes, spermatids and spermatozoa. Immunoblotting experiments demonstrate reaction with lamina polypeptides A, B and C of rat, with lamina polypeptide A of chicken, and with lamina polypeptides LI and LII of erythrocytes of the frog, Xenopus laevis. Antibody PKB8 does, however, not bind, on blotted polypeptides and on sections through ovaries, to the pore complex-lamina polypeptide of Mr 68000 present in Xenopus oocytes. These results reveal the existence of a common antigenic determinant in all three lamina polypeptides of mammals, in one lamina polypeptide of chicken and in two amphibian lamina polypeptides. The immunological data also indicate that, in Xenopus laevis, pore complex-lamina polypeptides of somatic cells and oocytes are distinct. The Mr 68000 protein of Xenopus oocytes is also different from polypeptides LI and LII of somatic Xenopus cells by tryptic peptide mapping. The results suggest that nuclear pore complex-lamina polypeptides represent a family of related polypeptides containing regions highly conserved during evolution and that these polypeptides can be differentially expressed in cells of at least one species, Xenopus laevis. PMID:6198191

Krohne, G; Debus, E; Osborn, M; Weber, K; Franke, W W

1984-01-01

280

Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73  

NASA Astrophysics Data System (ADS)

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

Zhang, Heng; Wu, Shengnan

2011-01-01

281

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

282

The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors  

SciTech Connect

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7{sub 39-98} localized mostly to the nucleus. The GST-11E7 and GST-11cE7{sub 39-98} were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona, E-mail: moroianu@bc.ed

2010-11-10

283

Atypical I?B proteinsnuclear modulators of NF-?B signaling  

PubMed Central

Nuclear factor ?B (NF-?B) controls a multitude of physiological processes such as cell differentiation, cytokine expression, survival and proliferation. Since NF-?B governs embryogenesis, tissue homeostasis and the functions of innate and adaptive immune cells it represents one of the most important and versatile signaling networks known. Its activity is regulated via the inhibitors of NF-?B signaling, the I?B proteins. Classical I?Bs, like the prototypical protein I?B?, sequester NF-?B transcription factors in the cytoplasm by masking of their nuclear localization signals (NLS). Thus, binding of NF-?B to the DNA is inhibited. The accessibility of the NLS is controlled via the degradation of I?B?. Phosphorylation of the conserved serine residues 32 and 36 leads to polyubiquitination and subsequent proteasomal degradation. This process marks the central event of canonical NF-?B activation. Once their NLS is accessible, NF-?B transcription factors translocate into the nucleus, bind to the DNA and regulate the transcription of their respective target genes. Several studies described a distinct group of atypical I?B proteins, referred to as the BCL-3 subfamily. Those atypical I?Bs show entirely different sub-cellular localizations, activation kinetics and an unexpected functional diversity. First of all, their interaction with NF-?B transcription factors takes place in the nucleus in contrast to classical I?Bs, whose binding to NF-?B predominantly occurs in the cytoplasm. Secondly, atypical I?Bs are strongly induced after NF-?B activation, for example by LPS and IL-1? stimulation or triggering of B cell and T cell antigen receptors, but are not degraded in the first place like their conventional relatives. Finally, the interaction of atypical I?Bs with DNA-associated NF-?B transcription factors can further enhance or diminish their transcriptional activity. Thus, they do not exclusively act as inhibitors of NF-?B activity. The capacity to modulate NF-?B transcription either positively or negatively, represents their most important and unique mechanistic difference to classical I?Bs. Several reports revealed the importance of atypical I?B proteins for immune homeostasis and the severe consequences following their loss of function. This review summarizes insights into the physiological processes regulated by this protein class and the relevance of atypical I?B functioning. PMID:23578005

2013-01-01

284

Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5  

SciTech Connect

Highlights: ? Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ? PRMT5 augments the EBNA2-dependent transcription. ? PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ? PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States)] [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

2013-01-18

285

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

286

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, Valérie

2014-01-01

287

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. PMID:25258565

Lee, Euna

2014-01-01

288

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress?  

PubMed Central

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress. PMID:19029246

Griffiths, Lyra M.; Swartzlander, Dan; Meadows, Kellen L.; Wilkinson, Keith D.; Corbett, Anita H.; Doetsch, Paul W.

2009-01-01

289

Dynamic compartmentalization of base excision repair proteins in response to nuclear and mitochondrial oxidative stress.  

PubMed

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress. PMID:19029246

Griffiths, Lyra M; Swartzlander, Dan; Meadows, Kellen L; Wilkinson, Keith D; Corbett, Anita H; Doetsch, Paul W

2009-02-01

290

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

PubMed Central

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

291

Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.  

PubMed

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light. PMID:25019686

Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

2014-01-01

292

N-Terminal Sequences from Autographa californica Nuclear Polyhedrosis Virus Envelope Proteins ODV-E66 and ODV-E25 are Sufficient to Direct Reporter Proteins to the Nuclear Envelope, Intranuclear Microvesicles and the Envelope of Occlusion Derived Virus  

Microsoft Academic Search

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to

Tao Hong; Max D. Summers; Sharon C. Braunagel

1997-01-01

293

Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2)  

PubMed Central

During influenza virus infection, viral ribonucleoproteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP defines an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a C- terminal domain which binds to the viral matrix protein M1. The 2.6 ? crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP–M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed. PMID:12970177

Akarsu, Hatice; Burmeister, Wilhelm P.; Petosa, Carlo; Petit, Isabelle; Müller, Christoph W.; Ruigrok, Rob W.H.; Baudin, Florence

2003-01-01

294

Association of hepatitis B virus polymerase with promyelocytic leukemia nuclear bodies mediated by the S100 family protein p11.  

PubMed

Hepatitis B virus (HBV) polymerase (Pol) interacts with cellular chaperone proteins and thereby performs multiple functions necessary for viral replication. Yeast two-hybrid analysis was applied to identify additional cellular targets required for HBV Pol function. HBV Pol interacted with S100A10 (p11), a Ca(2+)-modulated protein previously shown to bind to annexin II. The interaction between HBV Pol and p11 was confirmed by co-immunoprecipitation of the two proteins synthesized either in vitro or in transfected cells and by inhibition of the DNA polymerase activity of HBV Pol by p11. Immunofluorescence analysis of transfected human cell lines revealed that, although most HBV Pol and p11 was restricted to the cytoplasm, a small proportion of each protein colocalized as nuclear speckles; HBV Pol was not detected in the nucleus in the absence of p11. The HBV Pol-p11 nuclear speckles coincided with nuclear bodies containing the promyelocytic leukemia protein PML. Furthermore, the association of HBV Pol-p11 with PML was increased by exposure of cells to EGTA and inhibited by valinomycin. These results suggest a role for p11 in modulation of HBV Pol function and implicate PML nuclear bodies and intracellular Ca(2+) in viral replication. PMID:12767936

Choi, Juhyun; Chang, Jin-Sook; Song, Min-Sup; Ahn, Byung-Yoon; Park, Young; Lim, Dae-Sik; Han, Ye Sun

2003-06-13

295

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

296

Structure-function-folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights.  

PubMed

The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure-function-folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years. PMID:19805907

Mohan, P M Krishna; Hosur, Ramakrishna V

2009-09-01

297

Purification of ADP-Ribosylated Nuclear Proteins by Covalent Chromatography on Dihydroxyboryl Polyacrylamide Beads and their Characterization  

Microsoft Academic Search

Nuclear proteins modified by mono or poly ADP-ribosylation were selectively isolated and purified by covalent chromatography on a dihydroxyboryl polyacrylamide bead column that specifically interacts with cis-diol-containing compounds. From rat liver nuclei that had been incubated with NAD+, histones and some nonhistone proteins were extracted with 0.25 M HCl. Approximately 60% of the ADP-ribose incorporated into 20% trichloroacetic acid-precipitable material

Hiroto Okayama; Kunihiro Ueda; Osamu Hayaishi

1978-01-01

298

Developmentally essential protein flightless I is a nuclear receptor coactivator with actin binding activity.  

PubMed

Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins. PMID:14966289

Lee, Young-Ho; Campbell, Hugh D; Stallcup, Michael R

2004-03-01

299

Intermolecular masking of the HIV1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import  

Microsoft Academic Search

Background  The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral\\u000a transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition\\u000a of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin ?. However, it\\u000a remains unclear which nuclear import pathway(s) predominate

Lili Gu; Takahiro Tsuji; Ali Mohamed Jarboui; Geok P Yeo; Noreen Sheehy; William W Hall; Virginie W Gautier

2011-01-01

300

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

301

Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB.  

PubMed

Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher Wj

2015-03-01

302

Las1 Is an Essential Nuclear Protein Involved in Cell Morphogenesis and Cell Surface Growth  

PubMed Central

Saccharomyces cerevisiae mutations that cause a requirement for SSD1-v for viability were isolated, yielding one new gene, LAS1, and three previously identified genes, SIT4, BCK1/SLK1, and SMP3. Three of these genes, LAS1, SIT4, and BCK1/SLK1, encode proteins that have roles in bud formation or morphogenesis. LAS1 is essential and loss of LAS1 function causes the cells to arrest as 80% unbudded cells and 20% large budded cells that accumulate many vesicles at the mother-daughter neck. Overexpression of LAS1 results in extra cell surface projections in the mother cell, alterations in actin and SPA2 localization, and the accumulation of electron-dense structures along the periphery of both the mother cell and the bud. The nuclear localization of LAS1 suggests a role of LAS1 for regulating bud formation and morphogenesis via the expression of components that function directly in these processes. PMID:8582632

Doseff, A. I.; Arndt, K. T.

1995-01-01

303

A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein*  

PubMed Central

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1–P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3–P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection. PMID:22700958

Oksayan, Sibil; Wiltzer, Linda; Rowe, Caitlin L.; Blondel, Danielle; Jans, David A.; Moseley, Gregory W.

2012-01-01

304

SU-E-J-61: Electrodynamics and Nano-Scale Fluid Dynamics in Protein Localization of Nuclear Pore Complexes  

SciTech Connect

Purpose: To develop a simulation to catalyze a reevaluation of common assumptions about 3 dimensional diffusive processes and help cell biologists gain a more nuanced, intuitive understanding of the true physical hurdles of protein signaling cascades. Furthermore, to discuss the possibility of intracellular electrodynamics as a critical, unrecognized component of cellular biology and protein dynamics that is necessary for optimal information flow from the cell membrane to the nucleus. Methods: The Unity 3D gaming physics engine was used to build an accurate virtual scale model of the cytoplasm within a few hundred nanometers of the nuclear membrane. A cloud of simulated pERK proteins is controlled by the physics simulation, where diffusion is based on experimentally measured values and the electrodynamics are based on theoretical nano-fluid dynamics. The trajectories of pERK within the cytoplasm and through the 1250 nuclear pores on the nuclear surface is recorded and analyzed. Results: The simulation quickly demonstrates that pERKs moving solely by diffusion will rarely locate and come within capture distance of a nuclear pore. The addition of intracellular electrodynamics between charges on the nuclear pore complexes and on pERKs increases the number of successful translocations by allowing the electro-physical attractive effects to draw in pERKs from the cytoplasm. The effects of changes in intracellular shielding ion concentrations allowed for estimation of the “capture radius” under varying conditions. Conclusion: The simulation allows a shift in perspective that is paramount in attempting to communicate the scale and dynamics of intracellular protein cascade mechanics. This work has allowed researchers to more fully understand the parameters involved in intracellular electrodynamics, such as shielding anion concentration and protein charge. As these effects are still far below the spatial resolution of currently available measurement technology this simulation is invaluable to researchers to visualize and study current hypotheses concerning protein dynamics.

Cunningham, J; Gatenby, R [Moffitt Cancer Research Institute, Tampa, FL (United States)

2014-06-01

305

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; Houlné, Guy; Herzog, Etienne; Evrard, Jean-Luc; Baumberger, Nicolas; Erhardt, Mathieu; Nominé, Yves; Kieffer, Bruno; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2013-01-01

306

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

307

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

308

Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web  

PubMed Central

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin ?5 (IPOA5/kap?1), importin ?3 (IPO5/kap ?3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication. PMID:25485706

Levin, Aviad; Neufeldt, Christopher J.; Pang, Daniel; Wilson, Kristen; Loewen-Dobler, Darci; Joyce, Michael A.; Wozniak, Richard W.; Tyrrell, D. Lorne J

2014-01-01

309

Plasmodium falciparum Tudor Staphylococcal Nuclease interacting proteins suggest its role in nuclear as well as splicing processes.  

PubMed

Tudor Staphylococcal Nuclease (p100 or SND1), a member of the micronuclease family is a multifunctional protein that plays a key role(s) in transcription and splicing processes in many eukaryotic cells. PfTudor-SN, a Plasmodium homolog of the human p100 protein is a structurally conserved protein; however molecular details of its function are not yet understood. Our previous studies have shown that PfTudor-SN binds RNA and it is possible to selectively inhibit parasite growth by PfTudor-SN specific drugs. In the present study, we identified the molecular interactions between Plasmodium falciparum Tudor-SN and twelve Plasmodium proteins such as Histone h2A, SPT2 (a transcriptional regulator), a Cold-shock DNA binding protein in a bacterial two-hybrid screen. To get further insight into some of these interactions, we mapped the interaction domain in PfTudor-SN protein using the yeast two-hybrid system. Of these proteins, Plasmodium N-methyl-d-aspartate receptor associated protein, PfUbiquitin conjugating enzyme and Cold-shock DNA binding protein showed interaction with the SN domains of PfTudor-SN. Immuno-localization studies of the interacting proteins showed their presence predominantly in the nucleus, which inevitably suggests the molecular interactions between these proteins and PfTudor-SN. Furthermore, we also identified a molecular interaction between the Tudor domain of PfTudor-SN protein and Plasmodium spliceosomal Sm protein, PfSmD1 advocating the role of PfTudor-SN in the spliceosome assembly. Together, these results suggest multiple role(s) for PfTudor-SN protein mainly in nuclear and splicing processes at asexual blood stages of the malaria parasite. PMID:20713134

Hossain, Manzar J; Korde, Reshma; Singh, Prashant K; Kanodia, Shivani; Ranjan, Ravi; Ram, Geeta; Kalsey, Gaganjot Singh; Singh, Rita; Malhotra, Pawan

2010-11-15

310

A Polynomial-Time Algorithm for De Novo Protein Backbone Structure Determination from Nuclear Magnetic Resonance Data  

Microsoft Academic Search

We describe an efficient algorithm for protein backbone structure determination from solu- tion Nuclear Magnetic Resonance (NMR) data. A key feature of our algorithm is that it finds the conformation and orientation of secondary structure elements as well as the global fold in polynomial time. This is the first polynomial-time algorithm for de novo high-resolution biomacromolecular structure determination using experimentally

Lincong Wang; Ramgopal R. Mettu; Bruce Randall Donald

2006-01-01

311

Regulation of FE65 nuclear translocation and function by amyloid beta-protein precursor in osmotically stressed cells.  

PubMed

FE65, a neural adaptor protein, interacts with amyloid beta-protein precursor (APP) and is known to regulate amyloid beta generation from APP. FE65 also associates with nuclear proteins; however, its physiological function in the nucleus remains unclear. A fixed population of cytoplasmic FE65 is tethered to membranes by binding APP. This membrane-tethered FE65 is liberated from membranes by APP phosphorylation, which is facilitated by a stress-activated protein kinase in sorbitol-treated cells. Here we show that liberated FE65, which is distinct from "virgin" FE65 in the cytoplasm, translocates into the nucleus and accumulates in the nuclear matrix forming a patched structure. Targeting of FE65 into the nuclear matrix was suppressed by the APP intracellular domain fragment, which is generated by consecutive cleavages of APP. Thus, nuclear translocation of FE65 is under the regulation of APP. In the nucleus, FE65 induced gammaH2AX, which plays an important role in DNA repair as a cellular response by stress-damaged cells. These observations suggest that APP-regulated FE65 plays an important role in the early stress response of cells and that FE65 deregulated from APP induces apoptosis. PMID:18468999

Nakaya, Tadashi; Kawai, Tomoko; Suzuki, Toshiharu

2008-07-01

312

Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1.  

PubMed

Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUS(R495X) transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription. PMID:25274782

Tibshirani, Michael; Tradewell, Miranda L; Mattina, Katie R; Minotti, Sandra; Yang, Wencheng; Zhou, Hongru; Strong, Michael J; Hayward, Lawrence J; Durham, Heather D

2015-02-01

313

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

314

Purification of mouse MEP-1, a nuclear protein which binds to the metal regulatory elements of genes encoding metallothionein.  

PubMed Central

Metal regulatory elements (MREs) shared by metallothionein (MT) gene promoters are essential for metal induction of MT genes. MEP-1, a nuclear protein which binds to these elements has been purified from heavy metal-resistant mouse L cells using footprinting, Southwestern and UV cross-linking techniques to assay its binding activity. The purification scheme, starting from crude nuclear extracts, involved a combination of heparin-Sepharose and MRE-DNA affinity chromatography. The purified protein preparation showed a single polypeptide band of 108 kDa on polyacrylamide gel electrophoresis, and 2D-gel analyses revealed the presence of a protein species migrating as a single population of approximately 110 kDa. MEP-1 does not appear to be glycosylated since it eluted with the flow-through on a Wheat Germ Sepharose column. It was retained by a zinc-Chelating Sepharose column suggesting that amino acid residues (i.e., cysteine, histidine) which have an affinity for zinc ions are exposed on the protein surface. Binding studies with the purified protein indicated that it binds specifically to MRE sequences and that the binding can be abolished by a point mutation in the MRE core consensus sequence or by the addition of the chelating agent 1,10-phenanthroline. Binding activity can be restored by the addition of zinc ions to the chelated protein. These results suggest that MEP-1 is one of the major proteins interacting with MRE sequences. Images PMID:8479904

Labbé, S; Larouche, L; Mailhot, D; Séguin, C

1993-01-01

315

REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export.  

PubMed

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon-exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, we show that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, we conclude that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. Our results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1. PMID:12438415

Gatfield, David; Izaurralde, Elisa

2002-11-25

316

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

317

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

318

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

319

Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein.  

PubMed

Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS (also known as PY-NLS). Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis. PMID:21647358

Leemann-Zakaryan, Ruzanna P; Pahlich, Steffen; Grossenbacher, Doris; Gehring, Heinz

2011-01-01

320

Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein  

PubMed Central

Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS (also known as PY-NLS). Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis. PMID:21647358

Leemann-Zakaryan, Ruzanna P.; Pahlich, Steffen; Grossenbacher, Doris; Gehring, Heinz

2011-01-01

321

Growth Factor-induced p42\\/p44 MAPK Nuclear Translocation and Retention Requires Both MAPK Activation and Neosynthesis of Nuclear Anchoring Proteins  

Microsoft Academic Search

Mitogen-activated protein kinases (p42\\/p44 MAPK, also called Erk2 and Erk1) are key mediators of signal transduction from the cell surface to the nu- cleus. We have previously shown that the activation of p42\\/p44 MAPK required for transduction of mitogenic signaling is associated with a rapid nuclear transloca- tion of these kinases. However, the means by which p42 and p44 MAPK

Philippe Lenormand; Jean-Marc Brondello; Anne Brunet; Jacques Pouysségur

2010-01-01

322

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix  

SciTech Connect

Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, {beta}-sarcoglycan, {beta}-dystroglycan, {alpha}- and {beta}-syntrophin, {alpha}1- and {beta}-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, {beta}-dystroglycan, nNOS, {beta}-sarcoglycan, {alpha}/{beta} syntrophin, {alpha}1-dystrobrevin and {beta}-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, {beta}-dystroglycan and {beta}-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture.

Fuentes-Mera, Lizeth [Department of Genetics and Molecular Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida Instituto Politecnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F. (Mexico); Rodriguez-Munoz, Rafael [Department of Genetics and Molecular Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida Instituto Politecnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F. (Mexico); Gonzalez-Ramirez, Ricardo [Department of Genetics and Molecular Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida Instituto Politecnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F. (Mexico); Garcia-Sierra, Francisco [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida Instituto Politecnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F. (Mexico); Gonzalez, Everardo [Department of Genetics and Molecular Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida Instituto Politecnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F. (Mexico); Mornet, Dominique [Departement de Physiologie des Interactions (EA701), Institut de Biologie, Bvd Henry IV, 34060 Montpellier (France); Cisneros, Bulmaro [Department of Genetics and Molecular Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida Instituto Politecnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F. (Mexico)]. E-mail: bcisnero@cinvestav.mx

2006-10-01

323

Activation of nuclear polypeptide synthesis in rat liver cells in the case of inhibition of template synthesis of proteins. Contribution of nuclear polypeptide synthesis to the change in the chromatin structure  

SciTech Connect

The correlation between the activity of nuclear polypeptide synthesis (NPS) and template synthesis of proteins in rat liver cells was investigated. It was established that the NPS is activated under conditions of inhibition of protein synthesis in the cytoplasm. It was shown on model experiments that the systems of NPS and ADP-ribosylation of nuclear proteins compete in determining the general structure of chromatin: ADP-ribosylation promotes condensation, while NPS promotes decondensation of the chromatin.

Gutnikova, M.N.; Shevchenko, N.A.; Boikov, P.Ya.; Mitrokhin, Yu.I.; Todorov, I.N.

1986-06-10

324

KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei  

PubMed Central

Background Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. Methodology/Principal Findings We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. Conclusions/Significance The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod. PMID:21187923

Tan, Fu-Qing; Yang, Wan-Xi

2010-01-01

325

Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication  

SciTech Connect

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.

Lee, Changhee [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Hodgins, Douglas [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Calvert, Jay G. [Pfizer Animal Health, Kalamazoo, MI 49001 (United States); Welch, Siao-Kun W. [Pfizer Animal Health, Kalamazoo, MI 49001 (United States); Jolie, Rika [Pfizer Animal Health, Kalamazoo, MI 49001 (United States); Yoo, Dongwan [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada)]. E-mail: dyoo@uoguelph.ca

2006-03-01

326

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

327

Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran\\/TC4 GTPase mediates an essential function independent of nuclear protein import  

Microsoft Academic Search

The Ran\\/TC4 GTPase is required for the nu- clear accumulation of artificial karyophiles in perme- abilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorti- coid receptor-green fluorescent protein fusion (GR- GFP). Glucocorticoid receptor is cytosolic

Kimberly L. Carey; Stephanie A. Richards; Karen M. Lounsbury; Ian G. Macara

1996-01-01

328

The nuclear pore complex-associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly  

Microsoft Academic Search

he two yeast proteins Mlp1p and Mlp2p (homo- logues of the vertebrate protein Tpr) are filamentous proteins attached to the nuclear face of nuclear pore complexes. Here we perform a proteomic analysis, which reveals that the two Mlps have strikingly different interact- ing partners, testifying to their different roles within the cell. We find that Mlp2p binds directly to Spc110p,

Mario Niepel; Caterina Strambio-de-Castillia; Joseph Fasolo; Brian T. Chait; Michael P. Rout

2005-01-01

329

The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture  

PubMed Central

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE. PMID:25023515

Cain, Natalie E.; Tapley, Erin C.; McDonald, Kent L.; Cain, Benjamin M.

2014-01-01

330

The role of nuclear matrix proteins binding to matrix attachment regions (Mars) in prostate cancer cell differentiation.  

PubMed

In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy. PMID:22808207

Barboro, Paola; Repaci, Erica; D'Arrigo, Cristina; Balbi, Cecilia

2012-01-01

331

Development of a Radioiodinated Triazolopyrimidine Probe for Nuclear Medical Imaging of Fatty Acid Binding Protein 4  

PubMed Central

Fatty acid binding protein 4 (FABP4) is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki?=?44.5±9.8 nM, Kd?=?69.1±12.3 nM). The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection). The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging. PMID:24732569

Onoe, Satoru; Sampei, Sotaro; Kimura, Ikuo; Ono, Masahiro; Saji, Hideo

2014-01-01

332

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

Iowa Public Television. Explore More Project

2004-01-01

333

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

334

Hepatitis C virus non-structural 5A protein can enhance full-length core protein-induced nuclear factor-?B activation  

PubMed Central

AIM: To study the effects of hepatitis C virus (HCV) core and non-structural 5A (NS5A) proteins on nuclear factor-?B (NF-?B) activity for understanding their biological function on chronic hepatitis caused by HCV infection. METHODS: Luciferase assay was used to measure the activity of NF-?B in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF-?B-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF-?B/p65, NF-?B/p50, and inhibitor ?B-a (I?B-a). RESULTS: The wild-type core protein (C191) and its mutant segments (C173 and C158) could activate NF-?B in Huh7 cells only and activation caused by (C191) could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF-?B. The NF-?B activity was augmented due to the dissociation of NF-?B-I?B complex and the degradation of I?B-a. CONCLUSION:NF-?B is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF-?B activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection. PMID:16425412

Liao, Qing-Jiao; Ye, Lin-Bai; Timani, Khalid Amine; She, Ying-Long; Yang, Xiao-Jun; Ye, Li; Wu, Zheng-Hui

2005-01-01

335

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

336

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression  

PubMed Central

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP- bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis. PMID:8188741

1994-01-01

337

The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity  

PubMed Central

Background Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. Results We have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected. Conclusions Our results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals. PMID:15193161

Cikala, Mihai; Alexandrova, Olga; David, Charles N; Pröschel, Matthias; Stiening, Beate; Cramer, Patrick; Böttger, Angelika

2004-01-01

338

Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p.  

PubMed Central

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA. PMID:9545233

Senger, B; Simos, G; Bischoff, F R; Podtelejnikov, A; Mann, M; Hurt, E

1998-01-01

339

The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumour cell survival.  

PubMed

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide variety of human cancers, the retinoblastoma protein (Rb) has been shown to be overexpressed in colon cancers, which is linked to the anti-apoptotic function of the protein. However, the mechanisms by which Rb regulates apoptosis are yet to be fully elucidated. We have established that Rb interacts with the anti-apoptotic BAG-1 (Bcl-2 associated athanogene-1) protein, and that a decrease in nuclear localization of BAG-1 is detectable when the interaction between Rb and BAG-1 is disrupted by expression of the E7 viral oncoprotein. Interestingly, although reported as deregulated in colorectal cancers, we have found that BAG-1 expression is also altered in small adenomas, where its localization was found to be predominantly nuclear. In addition, we have established that maintenance of high nuclear BAG-1 in vitro increases the resistance of adenoma-derived cells to gamma-radiation-induced apoptosis. Our work suggests a novel function for Rb, involving modulation of the subcellular localization of BAG-1. We have found predominant nuclear BAG-1 localization in small adenomas, and suggest that BAG-1 may promote colorectal tumour cell survival by making colonic epithelial cells less sensitive to DNA damage. PMID:16042572

Clemo, N K; Arhel, N J; Barnes, J D; Baker, J; Moorghen, M; Packham, G K; Paraskeva, C; Williams, A C

2005-08-01

340

Characterization of a nuclear compartment shared by nuclear bodies applying ectopic protein expression and correlative light and electron microscopy  

Microsoft Academic Search

To investigate the accessibility of interphase nuclei for nuclear body-sized particles, we analyzed in cultured cells from human origin by correlative fluorescence and electron microscopy (EM) the bundle-formation of Xenopus-vimentin targeted to the nucleus via a nuclear localization signal (NLS). Moreover, we investigated the spatial relationship of speckles, Cajal bodies, and crystalline particles formed by Mx1 fused to yellow fluorescent

Karsten Richter; Michaela Reichenzeller; Sabine M. Goerisch; Ute Schmidt; Markus O. Scheuermann; Harald Herrmann; Peter. Lichter

2005-01-01

341

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

342

Nuclear localized protein-1 (Nulp1) increases cell death of human osteosarcoma cells and binds the X-linked inhibitor of apoptosis protein  

SciTech Connect

Nuclear localized protein-1 (Nulp1) is a recently identified gene expressed in mouse and human tissues particularly during embryonic development. Nulp1 belongs to the family of basic helix-loop-helix (bHLH) proteins that are important in development. The precise function of Nulp1 in cells is however not known. We observed that overexpression of Nulp1 induces a large increase in cell death of human osteosarcoma Saos2 cells with DNA fragmentation. In mouse N2A neuroblastoma cells Nulp1 affected cell proliferation and sensitized cells towards death induced by staurosporine. Staining using a novel antibody localized Nulp1 mainly to the cell nucleus and to some extent to the cytoplasm. Nulp1 binds the X-linked inhibitor of apoptosis protein (XIAP) and this interaction was increased during cell death. These results indicate that Nulp1 plays a role in cell death control and may influence tumor growth.

Steen, Hakan [Department of Neuroscience, Uppsala University, Biomedical Centre, Box 587, Husargatan 3, SE-75123 Uppsala (Sweden); Lindholm, Dan [Department of Neuroscience, Uppsala University, Biomedical Centre, Box 587, Husargatan 3, SE-75123 Uppsala (Sweden); Minerva Institute for Medical Research, Biomedicum Helsinki, Helsinki (Finland)], E-mail: dan.lindholm@neuro.uu.se

2008-02-08

343

Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells  

SciTech Connect

A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of (/sup 35/S)methionine-labelled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.

Celis, J.E.; Madsen, P.; Nielsen, S.; Ratz, G.P.; Lauridsen, J.B.; Celis, A.

1987-02-01

344

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities  

SciTech Connect

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.

Lin, W.-L. [Graduate Institute of Veterinary Microbiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Chien, M.-S. [Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Du, Y.-W.; Wu, P.-C. [Graduate Institute of Veterinary Microbiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Huang Chienjin [Graduate Institute of Veterinary Microbiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China)], E-mail: cjhuang@dragon.nchu.edu.tw

2009-02-20

345

Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1  

PubMed Central

Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies. PMID:25145264

Ishizuka, Akira; Hasegawa, Yuko; Ishida, Kentaro; Yanaka, Kaori; Nakagawa, Shinichi

2014-01-01

346

Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1.  

PubMed

Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies. PMID:25145264

Ishizuka, Akira; Hasegawa, Yuko; Ishida, Kentaro; Yanaka, Kaori; Nakagawa, Shinichi

2014-09-01

347

Protein  

NSDL National Science Digital Library

Protein structure: Primary protein structure is a sequence of amino acids. Secondary protein structure occurs when the amino acids in the sequence are linked by hydrogen bonds. Tertiary protein structure occurs when certain attractions are present between alpha helices and pleated sheets. Quaternary protein structure is a protein consisting of more than one amino acid chain.

Darryl Leja (National Human Genome Research Institute REV)

2005-04-04

348

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

349

The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels  

SciTech Connect

We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.

Hwang, Mi-Na [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of); Min, Chan-Hee [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of); Kim, Hyung Sik [College of Pharmacy, Pusan National University, Busan (Korea, Republic of); Lee, Ho [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of); Yoon, Kyong-Ah [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of); Park, Sung Yong [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of); Lee, Eun Sook [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of); Yoon, Sungpil [Research Institute, National Cancer Center, 809 Madu l-dong, Ilsan-gu, Goyang-si Gyeonggi-do 411-764 (Korea, Republic of)]. E-mail: yoons@ncc.re.kr

2007-08-24

350

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

PubMed

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. PMID:21605886

Sides, Mark D; Block, Gregory J; Shan, Bin; Esteves, Kyle C; Lin, Zhen; Flemington, Erik K; Lasky, Joseph A

2011-07-20

351

The latency-associated nuclear antigen, a multifunctional protein central to Kaposi’s sarcoma-associated herpesvirus latency  

PubMed Central

Latency-associated nuclear antigen (LANA) is encoded by the Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) open reading frame 73. LANA is expressed during latent KSHV infection of cells, including tumor cells, such as primary effusion lymphoma, KS and multicentric Castleman’s disease. Latently infected cells have multiple extrachromosomal copies of covalently closed circular KSHV genomes (episomes) that are stably maintained in proliferating cells. LANA’s best characterized function is that of mediating episome persistence. It does so by binding terminal repeat sequences to the chromosomal matrix, thus ensuring episome replication with each cell division and efficient DNA segregation to daughter nuclei after mitosis. To achieve these functions, LANA associates with different host cell proteins, including chromatin-associated proteins and proteins involved in DNA replication. In addition to episome maintenance, LANA has transcriptional regulatory effects and affects cell growth. LANA exerts these functions through interactions with different cell proteins. PMID:22122438

Ballestas, Mary E; Kaye, Kenneth M

2013-01-01

352

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

353

Changes in the two-dimensional proteome of the soluble fraction of nuclear proteins from Lepidium sativum root meristematic cells grown under clinorotation.  

PubMed

The changes in the fundamental biological processes of nuclear RNA transcription and splicing under altered gravity conditions are still unclear. The quantitative and qualitative characteristics of the proteins involved in nuclear RNA metabolism in control and under clinorotation were investigated by two-dimensional gel electrophoresis. We revealed firstly a decrease in the isoelectric point range of nuclear soluble proteins, which are known to be actively engaged in nuclear RNA metabolism, and a shortening in the molecular weight range of them under clinorotation. Moreover, minor and major proteins in clinorotated samples had decreased optical densities in comparison with control ones. Our results are in agreement with the hypothesis that a rearrangement of the pattern of nuclear proteins involved in gene expression processes occurs in seedlings grown and developed in altered gravity. PMID:18372723

Sobol, M A; González-Camacho, F; Kordyum, E L; Medina, F J

2007-07-01

354

Nuclear protein I{kappa}B-{zeta} inhibits the activity of STAT3  

SciTech Connect

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein I{kappa}B-{zeta} that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of I{kappa}B-{zeta}. Overexpression of I{kappa}B-{zeta} inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that I{kappa}B-{zeta} is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-{kappa}B signaling pathway inhibits the activation of STAT3.

Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China)] [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China); Chang, Zhijie [Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing 100084 (China)] [Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing 100084 (China); Wang, Jian, E-mail: wangjian@nic.bmi.ac.cn [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China)] [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China); Yang, Xiaoming, E-mail: xmyang2@nic.bmi.ac.cn [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China)] [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China); He, Fuchu, E-mail: hefc@nic.bmi.ac.cn [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China) [State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850 (China); Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China)

2009-09-18

355

Nuclear factors specifically bind to upstream sequences of a Xenopus laevis ribosomal protein gene promoter.  

PubMed Central

The upstream region of the Xenopus laevis L14 ribosomal protein gene was deleted starting from the 5' extremity in order to define the promoter length necessary to express a linked reporter CAT gene. The functional analysis indicated that a sequence located between -63 and -49 from the capsite is important for an efficient promoter activity. Band shift and ExoIII protection assays evidenced the binding to this region of a factor, called XrpFI, present in the crude nuclear extract from X.laevis oocytes. Methylation interference analysis localized the contacts in the G residues belonging to a short box, 5' CTTCC 3', positioned between -53 and -49 from the capsite. An additional factor, XrpFII, makes contacts with the sequence 5'GCCTGTTCGCC 3' located between -27 and -17 from the capsite. The deletion mutant still containing this sequence is poorly transcribed, but resumes activity when a short fragment containing the binding site for factor XrpFI is cloned in an upstream position. Images PMID:2682523

Carnevali, F; La Porta, C; Ilardi, V; Beccari, E

1989-01-01

356

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

357

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, Silvère M.; Raz, Vered; ‘t Hoen, Peter A. C.

2012-01-01

358

Phylogenetic analysis of arthropods using two nuclear protein-encoding genes supports a crustacean + hexapod clade.  

PubMed Central

Recent phylogenetic analyses using molecular data suggest that hexapods are more closely related to crustaceans than to myriapods, a result that conflicts with long-held morphology-based hypotheses. Here we contribute additional information to this debate by conducting phylogenetic analyses on two nuclear protein-encoding genes, elongation factor-1 alpha (EF-1 alpha) and the largest subunit of RNA polymerase II (Pol II), from an extensive sample of arthropod taxa. Results were obtained from two data sets. One data set comprised 1092 nucleotides (364 amino acids) of EF-1 alpha and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF-1 alpha fragment and an expanded 1038-nucleotide (346-amino-acid) sample of Pol II from 17 arthropod taxa. Results from maximum-parsimony and maximum-likelihood analyses strongly supported the existence of a Crustacea + Hexapoda clade (Pancrustacea) over a Myriapoda + Hexapoda clade (Atelocerata). The apparent incompatibility between the molecule-based Pancrustacea hypothesis and morphology-based Atelocerata hypothesis is discussed. PMID:10874751

Shultz, J W; Regier, J C

2000-01-01

359

31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes.  

PubMed Central

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component. PMID:6120013

Yeagle, P L

1982-01-01

360

Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1?) docking protein  

PubMed Central

Summary The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and II?. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway. PMID:23213405

Ferrar, Tony; Chamousset, Delphine; De Wever, Veerle; Nimick, Mhairi; Andersen, Jens; Trinkle-Mulcahy, Laura; Moorhead, Greg B. G.

2012-01-01

361

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

362

A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana.  

PubMed Central

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes. PMID:9736608

Meurer, J; Plücken, H; Kowallik, K V; Westhoff, P

1998-01-01

363

Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal  

SciTech Connect

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ({sup 211}KKYK{sup 214}) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to {beta}-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.

Pasdeloup, David [Unite Mixte de Virologie Moleculaire et Structurale UMR2472 CNRS, UMR1157 INRA, 91198 Gif sur Yvette Cedex (France); Poisson, Nicolas [Unite Mixte de Virologie Moleculaire et Structurale UMR2472 CNRS, UMR1157 INRA, 91198 Gif sur Yvette Cedex (France); Raux, Helene [Unite Mixte de Virologie Moleculaire et Structurale UMR2472 CNRS, UMR1157 INRA, 91198 Gif sur Yvette Cedex (France); Gaudin, Yves [Unite Mixte de Virologie Moleculaire et Structurale UMR2472 CNRS, UMR1157 INRA, 91198 Gif sur Yvette Cedex (France); Ruigrok, Rob W.H. [Laboratoire de Virologie Moleculaire et Structurale, FRE 2854 CNRS-UJF, c/o EMBL, BP 181, 38042 Grenoble Cedex 9 (France); Blondel, Danielle [Unite Mixte de Virologie Moleculaire et Structurale UMR2472 CNRS, UMR1157 INRA, 91198 Gif sur Yvette Cedex (France)]. E-mail: danielle.blondel@vms.cnrs-gif.fr

2005-04-10

364

Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data.  

PubMed

To test whether distances derived from paramagnetic broadening of (15)N heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of (15)N-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass approximately 45-50 kDa. Distance-dependent line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions. The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in (15)N-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 A (1.7 A for secondary structure elements). The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methyl-protonated, highly deuterated samples) improved the quality of calculated structures. In addition, the combination of the two methods simulated from a crystal structure of an all alpha-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR. PMID:10820006

Battiste, J L; Wagner, G

2000-05-01

365

Molecular cloning of cDNA for the nuclear ribonucleoprotein particle C proteins: a conserved gene family.  

PubMed Central

The C proteins, C1 and C2, are major constituents of the heterogeneous nuclear RNA (hnRNA) ribonucleoprotein (hnRNP) complex in vertebrates. C1 and C2 are antigenically related phosphoproteins that are in contact with hnRNA in intact cells and bind to RNA tightly in vitro. A cDNA clone for the C proteins was isolated by immunological screening of a human lambda gt11 expression vector cDNA library with monoclonal antibodies. The lacZ-cDNA fusion protein is recognized by two different anti-C protein monoclonal antibodies. HeLa cell mRNA that was hybrid-selected with the cDNA clone (1.1 kilobases long) was translated in vitro and yielded both the C1 and C2 proteins (41 and 43 kDa, respectively). RNA blot analysis showed strong hybridization to two polyadenylylated transcripts, of about 1.4 kb and 1.9 kb, in human cells. Genomic DNA blot analysis showed multiple hybridizing restriction fragments in human and mouse, and homologous DNA sequences are found across eukaryotes from human to yeast. These findings suggest that the sequences encoding the hnRNP C proteins are members of a conserved gene family and they open the way for detailed molecular and genetic studies of these proteins. Images PMID:3457372

Nakagawa, T Y; Swanson, M S; Wold, B J; Dreyfuss, G

1986-01-01

366

Leishmania major telomerase TERT protein has a nuclear/mitochondrial eclipsed distribution that is affected by oxidative stress.  

PubMed

In its canonical role the reverse transcriptase telomerase recovers the telomeric repeats that are lost during DNA replication. Other locations and activities have been recently described for the telomerase protein subunit TERT in mammalian cells. In the present work, using biochemistry, molecular biology, and electron microscopy techniques, we found that in the human parasite Leishmania major, TERT (and telomerase activity) shared locations between the nuclear, mitochondrial, and cytoplasmic compartments. Also, some telomerase activity and TERT protein could be found in ? 100-nm nanovesicles. In the mitochondrial compartment, TERT appears to be mainly associated with the kinetoplast DNA. When Leishmania cells were exposed to H2O2, TERT changed its relative abundance and activity between the nuclear and mitochondrial compartments, with the majority of activity residing in the mitochondrion. Finally, overexpression of TERT in Leishmania transfected cells not only increased the parasitic cell growth rate but also increased their resistance to oxidative stress. PMID:25312950

Campelo, Riward; Díaz Lozano, Isabel; Figarella, Katherine; Osuna, Antonio; Ramírez, José Luis

2015-01-01

367

PSBP-DOMAIN PROTEIN1, a Nuclear-Encoded Thylakoid Lumenal Protein, Is Essential for Photosystem I Assembly in Arabidopsis[W  

PubMed Central

To gain insights into the molecular details of photosystem I (PSI) biogenesis, we characterized the PsbP-domain protein1 (ppd1) mutant of Arabidopsis thaliana that specifically lacks PSI activity. Deletion of PPD1 results in an inability of the mutant to grow photoautotrophically and a specific loss of the stable PSI complex. Unaltered transcription and translation of plastid-encoded PSI genes indicate that PPD1 acts at the posttranslational level. In vivo protein labeling experiments reveal that the rate of synthesis of PSI reaction center proteins PsaA/B in ppd1 is comparable to that of wild-type plants, whereas the rate of turnover of PsaA/B proteins is higher in ppd1 than in wild-type plants. With increasing leaf age, PPD1 content decreases considerably, while PSI content remains constant. PPD1 is a nuclear-encoded thylakoid lumenal protein and is associated with PSI but is not an integral subunit of PSI. Biochemical and molecular analyses reveal that PPD1 interacts directly and specifically with PsaB and PsaA. Yeast two-hybrid experiments show that PPD1 interacts with some lumenal loops of PsaB and PsaA. Our results suggest that PPD1 is a PSI assembly factor that assists the proper folding and integration of PsaB and PsaA into the thylakoid membrane. PMID:23221595

Liu, Jun; Yang, Huixia; Lu, Qingtao; Wen, Xiaogang; Chen, Fan; Peng, Lianwei; Zhang, Lixin; Lu, Congming

2012-01-01

368

Seminal plasma proteins in prostatic carcinoma: increased nuclear semenogelin I expression is a predictor of biochemical recurrence after radical prostatectomy.  

PubMed

Semenogelins and eppin are seminal plasma proteins that form a complex and inhibit sperm motility. However, the role of these proteins in prostate cancer is poorly understood. We immunohistochemically stained for semenogelins I and II and eppin in 291 radical prostatectomy specimens. We then evaluated the association between their expressions in nuclei, cytoplasms, or intraluminal secretions of benign/high-grade prostatic intraepithelial neoplasia/carcinoma cells and clinicopathologic profile available for our patient cohort. Stains were positive in 32%/77%/84% (nuclear semenogelin I), 87%/94%/84% (nuclear semenogelin II), 56%/64%/37% (nuclear eppin), 7%/15%/11% (cytoplasmic semenogelin I), 6%/11%/9% (cytoplasmic semenogelin II), 68%/74%/95% (cytoplasmic eppin), 97%/98%/13% (secreted semenogelin I), 98%/97%/11% (secreted semenogelin II), and 97%/98%/48% (secreted eppin) of benign/prostatic intraepithelial neoplasia/carcinoma, respectively. The levels of nuclear semenogelin I/cytoplasmic eppin were significantly higher in carcinoma than in benign (P<.001/P<.001) or prostatic intraepithelial neoplasia (P<.001/P<.001) and in prostatic intraepithelial neoplasia than in benign (P<.001/P=.006). Significantly higher nuclear semenogelin II expression was found in prostatic intraepithelial neoplasia than in benign (P<.001) or carcinoma (P<.001). Significantly lower nuclear eppin expression was seen in carcinoma than in benign (P<.001) or prostatic intraepithelial neoplasia (P<.001). Secreted semenogelin I, secreted semenogelin II, and secreted eppin were all significantly lower in carcinoma than in benign (P<.001) or prostatic intraepithelial neoplasia (P<.001). There were no statistically significant correlations between each stain and clinicopathologic features except significantly lower nuclear eppin expression in Gleason score 8 or higher tumors. Kaplan-Meier and log-rank tests further revealed that patients with nuclear semenogelin I-positive tumor had a significantly higher risk for biochemical recurrence (P=.046). Multivariate Cox model showed a trend toward significance (P=.093) in nuclear semenogelin I positivity as an independent predictor for recurrence. These results suggest that nuclear semenogelin I expression could be a reliable prognosticator in men who undergo radical prostatectomy. PMID:22617231

Izumi, Koji; Li, Yi; Zheng, Yichun; Gordetsky, Jennifer; Yao, Jorge L; Miyamoto, Hiroshi

2012-11-01

369

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

370

Activation of Nuclear Factor-?B and Correlation with Elevated Plasma C-reactive Protein in Patients with Unstable Angina  

Microsoft Academic Search

Background. Unstable coronary syndromes are currently believed to be caused by rupture of an atherosclerotic plaque due to local events, which may be of general inflammatory etiology. There is increasing evidence that nuclear factor-?B (NF-?B) is a key transcription factor in controlling gene expression concerning inflammatory response, and that plasma concentrations of C-reactive protein (CRP) is a sensitive marker of

Jian-Jun Li; Chun-Hong Fang; Ming-Zhe Chen; Xin Chen; Stephen W. L Lee

2004-01-01

371

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

372

The Unfolded Protein Response Transducer Ire1p Contains a Nuclear Localization Sequence Recognized by Multiple Importins  

Microsoft Academic Search

The Ire1p transmembrane receptor kinase\\/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of

Laurence Goffin; Sadanand Vodala; Christine Fraser; Joanne Ryan; Mark Timms; Sarina Meusburger; Bruno Catimel; Edouard C. Nice; Pamela A. Silver; Chong-Yun Xiao; David A. Jans; Mary-Jane H. Gething

2006-01-01

373

Evolution \\/ Évolution 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, amelogenin

Nicolas Vidal; S. Blair Hedges

374

Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear envelope proteins in relation to synaptonemal complex attachment sites.  

PubMed

The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs. PMID:10198069

Alsheimer, M; von Glasenapp, E; Hock, R; Benavente, R

1999-04-01

375

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

376

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

377