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Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin  

SciTech Connect

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

Demura, T.; Driscoll, W.J.; Lee, Y.C.; Strott, C.A. (National Institute of Child Health and Human Development, Bethesda, MD (USA))



A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors  

PubMed Central

The ventromedial hypothalamus (VMH) plays a central role in the regulation of the female reproductive behavior lordosis, a behavior dependent upon the sequential activation of receptors for the ovarian steroid hormones estradiol (E) and progesterone (P). These receptors function as transcription factors to alter the expression of target genes. To discover behaviorally relevant genes targeted by E and P in the VMH, we used the differential display PCR to identify messenger RNAs that are differentially expressed in the hypothalamus of ovariectomized (ovx) rats treated with E alone compared with ovariectomized rats treated with E and P. We show here that one interesting mRNA within the hypothalamus that is repressed by P after E priming encodes the protein 25-Dx, the rat homolog of the human membrane-associated P-binding protein Hpr6.6. Neurons in the brain containing the highest levels of 25-Dx are located in several nuclei of the basal forebrain, including the VMH. 25-Dx expression is also higher in the hypothalamus of female P receptor “knockout” mice than in their wild-type littermates. These findings suggest a mechanism in which the activation of nuclear P receptor represses expression of a membrane P receptor, 25-Dx, during lordosis facilitation.

Krebs, Christopher J.; Jarvis, Erich D.; Chan, Johnny; Lydon, John P.; Ogawa, Sonoko; Pfaff, Donald W.



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.



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



Properties of proteins binding plasma progesterone in pregnant Cape porcupines (Hystrix africaeaustralis).  


The properties of progesterone-binding proteins in plasma of pregnant Cape porcupines were investigated using radiolabelled progesterone and either progesterone or cortisol as competing ligands as well as native plasma and heated (60 degrees C for 30 min) plasma. The results demonstrated that plasma from pregnant porcupines contains corticosteroid-binding globulin, but that it constitutes a significant portion of plasma progesterone-binding proteins only during the early stages of pregnancy. Corticosteroid-binding globulin of porcupines appears to be as heat labile as that of guinea-pigs. Concentrations of progesterone-binding proteins in plasma increased during pregnancy to reach concentrations at the eleventh week that were 25 times higher than those of progesterone; concentrations increased significantly (r2 = 0.88) with the increase in progesterone concentration. The results indicate that plasma progesterone-binding proteins in Cape porcupines (Old World hystricomorph) are similar in composition to those in guinea-pigs (New World hystricomorph). PMID:1432942

Louw, A I; van Wyk, V; van Aarde, R J



Nuclear protein extraction from frozen porcine myocardium.  


Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5±0.7% of total protein; mean±SE, n=9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofilament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosine phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the ?-agonist dobutamine. Upon treatment, PY-STAT3 increased 30.2±8.5-fold in total homogenates, but only 6.9±2.1-fold (n=4, P=0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks. PMID:21061196

Kuster, Diederik W D; Merkus, Daphne; Jorna, Huub J J; Dekkers, Dick H W; Duncker, Dirk J; Verhoeven, Adrie J M



Targeting proteins to the plant nuclear envelope.  


The nuclear envelope and the nuclear pore are important structures that both separate and selectively connect the nucleoplasm and the cytoplasm. The requirements for specific targeting of proteins to the plant nuclear envelope and nuclear pore are poorly understood. How are transmembrane-domain proteins sorted to the nuclear envelope and nuclear pore membranes? What protein-protein interactions are involved in associating other proteins to the nuclear pore? Are there plant-specific aspects to these processes? We are using the case of the nuclear pore-associated Ran-cycle component RanGAP (Ran GTPase-activating protein) to address these fundamental questions. Plant RanGAP is targeted to the nuclear pore by a plant-specific mechanism involving two families of nuclear pore-associated proteins [WIP (WPP-domain-interacting protein) and WIT (WPP-domain-interacting tail-anchored protein)] not found outside the land plant lineage. One protein family (WIP or WIT) is sufficient for RanGAP targeting in differentiated root cells, whereas both families are necessary in meristematic cells. A C-terminal predicted transmembrane domain is sufficient for targeting WIP proteins to the nuclear envelope. Nuclear-envelope targeting of WIT proteins requires a coiled-coil domain and is facilitated by HSC70 (heat-shock cognate 70 stress protein) chaperones and a class of plant-specific proteins resembling the RanGAP-targeting domain (WPP proteins). Taken together, this sheds the first light on the requirements and interdependences of nuclear envelope and nuclear pore targeting in land plants. PMID:20491658

Meier, Iris; Zhou, Xiao; Brkljaci?, Jelena; Rose, Annkatrin; Zhao, Qiao; Xu, Xianfeng Morgan



Nuclear pore proteins and cancer.  


Nucleocytoplasmic trafficking of macromolecules, a highly specific and tightly regulated process, occurs exclusively through the nuclear pore complex. This immense structure is assembled from approximately 30 proteins, termed nucleoporins. Here we discuss the four nucleoporins that have been linked to cancers, either through elevated expression in tumors (Nup88) or through involvement in chromosomal translocations that encode chimeric fusion proteins (Tpr, Nup98, Nup214). In each case we consider the normal function of the nucleoporin and its translocation partners, as well as what is known about their mechanistic contributions to carcinogenesis, particularly in leukemias. Studies of nucleoporin-linked cancers have revealed novel mechanisms of oncogenesis and in the future, should continue to expand our understanding of cancer biology. PMID:19577736

Xu, Songli; Powers, Maureen A



Reassembling proteins and chaperones in human nuclear matrix protein fractions.  


To detect putative filament forming components, nuclear matrix proteins were searched for proteins extensively reassembling from urea solution. Eight proteins, ubiquitously occurring in various human cell types, but not apparent in the cytosol, were registered by means of two-dimensional gel electrophoresis. They consisted of a protein exhibiting a novel amino acid sequence; of nuclear lamin B2, RbAp46, and RbAp48; and of four as yet unknown proteins. Furthermore, partial sequencing, mass spectrometry, and immunodetection of proteins demonstrated the presence of molecular chaperones and protein folding catalysts in the nuclear matrix fractions. In addition to a TCP-1-related protein, certain members of the heat shock, PDI, and calreticulin family of proteins were detected. On the basis of the absence of several other heat shock proteins in the nuclear matrix fraction, a general contamination by cytoplasmic chaperones appears unlikely. PMID:10404385

Gerner, C; Holzmann, K; Meissner, M; Gotzmann, J; Grimm, R; Sauermann, G



GAPDH mediates nitrosylation of nuclear proteins.  


S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction. PMID:20972425

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



GAPDH Mediates Nitrosylation of Nuclear Proteins  

PubMed Central

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

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



Nuclear matrix proteins and hereditary diseases.  


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

Sjakste, N; Sjakste, T



Inner nuclear membrane proteins: functions and targeting  

Microsoft Academic Search

:   We summarize the properties of integral membrane proteins that reside in the inner nuclear membrane, including lamin B receptor\\u000a (LBR), lamina-associated polypeptide (LAP) 1, LAP2, emerin, MAN1 and nurim. Most of these proteins interact with lamins and\\u000a chromatin. Some data also suggest more speculative functions such as gene regulation and possibly sterol metabolism. Mutations\\u000a in emerin and nuclear lamins

L. Holmer; H. J. Worman



Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells  

PubMed Central

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

Morinaga, Ayako; Burroughs, Alexander Maxwell; Kawaji, Hideya; Kubosaki, Atsutaka; Kimura, Ryuichiro; Tagata, Maiko; Ino, Yoko; Hirano, Hisashi; Chiba, Joe; Suzuki, Harukazu; Carninci, Piero; Hayashizaki, Yoshihide



Nuclear Export of Proteins and RNA  

Microsoft Academic Search

Several nuclear export receptors that facilitate the export of proteins and small RNAs from the nucleus to the cytoplasm have\\u000a been functionally characterized in Arabidopsis thaliana in the past few years. With the specific cargo molecules they transport, the export receptors supply the cytoplasm with information,\\u000a resulting in changes in cellular events. In this way, nuclear export receptors contribute to

Thomas Merkle


Nuclear variants of bone morphogenetic proteins  

Microsoft Academic Search

BACKGROUND: Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5\\/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. RESULTS: In all three proteins,

Jenny E Felin; Jaime L Mayo; Trina J Loos; J Daniel Jensen; Daniel K Sperry; Stephanie L Gaufin; Christopher A Meinhart; Jennie B Moss; Laura C Bridgewater



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


An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Bürglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non-nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope. PMID:3097026

Newmeyer, D D; Finlay, D R; Forbes, D J



Intrinsic and extrinsic negative regulators of nuclear protein transport processes  

PubMed Central

The nuclear–cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclear transport processes have been observed in stressed cells, which would change gene expressions. Some viruses interfere with nuclear transport in host cells to evade immune defense. Moreover, certain transport factors negatively regulate nuclear protein transport in cells. Understanding the regulatory mechanisms of nuclear–cytoplasmic trafficking not only provides important information about cellular processes, but also is of use for developing specific inhibitors for transport pathways.

Sekimoto, Toshihiro; Yoneda, Yoshihiro



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.

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



The nuclear envelope LEM-domain protein emerin.  


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



An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport.  


Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery. PMID:11483521

de Bruyn Kops, A; Guthrie, C



Alternative nuclear transport for cellular protein quality control  

PubMed Central

Herpesvirus capsids traverse the nuclear envelope by utilizing an unusual export pathway termed nuclear egress. In this process, the viral capsid is delivered into the perinuclear space, producing a vesicular intermediate after fission. After fusion with the outer nuclear membrane, the naked capsid is released into the cytosol. A recent study now suggests that this pathway might be an endogenous cellular pathway, co-opted by viruses, that serves to transport cellular cargo exceeding the size limit imposed by the nuclear pore complex. We propose that one function of this pathway is to transport nuclear protein aggregates to the cytosolic autophagy machinery. Our model has implications for our understanding of laminopathies and related diseases affecting proteins residing at the inner nuclear membrane and nuclear lamina.

Rose, April; Schlieker, Christian



Nuclear transport of influenza virus polymerase PA protein.  


The subcellular distribution of influenza polymerase PA subunit has been studied using a SV40-recombinant virus (SVPA76), which allows the expression and accumulation of this protein in COS-1 cells. In contrast to the complete nuclear localization observed for the PA subunit several hours after influenza virus infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA protein accumulated either in the nucleus, in the cytoplasm or was distributed throughout the cell. When cells were infected with the SVPA76 recombinant and superinfected with influenza virus, a clear increase in the proportion of cells showing nuclear localization of the PA protein was observed, suggesting that some trans-factor may be required to allow complete nuclear accumulation of the protein. Double infections using SVPA76 recombinant and either SVPB1 or SVNS recombinant viruses showed a complete correlation between expression of polymerase PB1 subunit or NS1 protein and nuclear localization of polymerase PA subunit. However, no such correlation was observed in the double infections of SVPA76 and SVNP recombinants. These results suggest that polymerase PB1 subunit and the non-structural proteins could be involved in the nuclear targeting or nuclear retention of influenza polymerase PA protein. PMID:1320800

Nieto, A; de la Luna, S; Bárcena, J; Portela, A; Valcárcel, J; Melero, J A; Ortín, J



Prediction and screening of nuclear targeting proteins with nuclear localization signals in Helicobacter pylori.  


Host cell pathology induced by nuclear targeting of bacterial proteins has recently been identified as a pathogenic mechanism of bacteria. However, very few bacterial proteins were identified to target the nuclei of host cells. This study was designed to screen nuclear targeting proteins with nuclear localization signals (NLSs) in Helicobacter pylori using a combination of bioinformatic analysis and the Gateway recombinational cloning system. Forty-nine functional or hypothetical proteins were predicted to carry the putative NLSs among 1570 open reading frames (ORFs) of H. pylori 26695. Entire sets of 49 H. pylori ORFs were cloned for the generation of green fluorescent protein-tagged proteins using the Gateway recombinational cloning system. Twenty-six H. pylori proteins with the putative NLSs were found to target in the nuclei of COS-7 cells, whereas 23 were localized in the cytoplasm of host cells. Deletion of NLS sequences from four selected nuclear targeting proteins, urease subunit A, Omp18, secreted protein involved in flagellar motility, and response regulator, resulted in cytoplasmic localization of these mutant proteins. In conclusion, a combination of bioinformatic analysis and the Gateway cloning system was shown to be a useful tool for large-scale screening of nuclear targeting proteins with NLSs in H. pylori, which can be used to better understand the H. pylori-directed host cell pathology. PMID:23079023

Lee, Jung Hwa; Jun, So Hyun; Baik, Seung Chul; Kim, Deok Ryong; Park, Jae-Yong; Lee, Yong Seok; Choi, Chul Hee; Lee, Je Chul



Biochemical and immunological characterization of pea nuclear intermediate filament proteins  

Microsoft Academic Search

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea ( Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric

Sonal S. D. Blumenthal; Gregory B. Clark; Stanley J. Roux



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:; 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:



Estradiol binding to nuclear matrix protein of pig adrenal cortex  

SciTech Connect

Binding of TH-estradiol can be shown in vitro after incubation with purified washed nuclei of sow adrenal cortex or with the insoluble nuclear matrix protein isolated from nuclei. The procedure modified after Berezney and Coffey treated washed nuclei sequentially with 1% Triton-X100, DNase, RNase and 2M NaCl to give an insoluble nuclear matrix protein preparation in which most of the phospholipid, DNA, RNA and protein was removed. Reagents were added to 10 mM Tris buffer containing 1 mM phenylmethyl sulfonyl fluoride, dithiothreitol and 0.2 mM or 5.0 mM MgCl2. Each treatment and washes were centrifuged at 4C. Suspensions of nuclei and nuclear matrix protein were incubated at 4C for 24 hrs. with 0.25 to 3.0 ng of TH-estradiol in 0.5 ml 10 mM Tris buffer with 5 mM MgCl2. Scatchard analysis of binding in duplicate or triplicate tubes with or without excess unlabeled estradiol gave specific binding for sow adrenal nuclei and for nuclear matrix protein. Total binding sites varied between 780 to 1380 fmoles/mg protein. Estradiol binding was not shown in the fetal adrenal matrix nor in mitochondria. Noncompetitive controls included progesterone and pregnenolone. Nuclear matrix protein binding of estradiol may have significance in functional or morphological changes of the adrenal cortex in fetal, neonatal, or pubertal development.

Ungar, F.; Johnson, S.R.; Johnston, J.A.



A sequence motif conserved in diverse nuclear proteins identifies a protein interaction domain utilised for nuclear targeting by human TFIIS  

PubMed Central

The three structural domains of transcription elongation factor TFIIS are conserved from yeast to human. Although the N-terminal domain is not needed for transcriptional activity, a similar sequence has been identified previously in other transcription factors. We found this conserved sequence, the LW motif, in another three human proteins that are predominantly nuclear localized. We investigated two examples to determine whether the LW motif is actually a dedicated nuclear targeting signal. However, in one of the newly identified proteins, hIWS1 (human Iws1), a region containing classic nuclear localization signals (NLS) rather than the LW motif was necessary and sufficient for nuclear targeting in HeLa cells. In contrast, human TFIIS does not possess an NLS and only constructs containing the LW motif were efficiently targeted to nuclei. Moreover, mutations in the motif could cause cytoplasmic accumulation of TFIIS and enabled a structure/function assay for the domain based on the efficiency of nuclear targeting. Finally, GST pull-down assays showed that the LW motif is part of a protein-binding domain. We suggest that the targeting role the LW motif plays in TFIIS arises from its more general function as a protein interaction domain, enabling TFIIS to bind a carrier protein(s) that accomplishes nuclear import.

Ling, Yan; Smith, Abigail J.; Morgan, Garry T.



Nuclear localization of the zebrafish tight junction protein nagie oko.  


The tight junctions-associated MAGUK protein nagie oko is closely related to Drosophila Stardust, mouse protein associated with lin-seven 1 (Pals1), and human MAGUK p55 subfamily member 5 (Mpp5). As a component of the evolutionarily conserved Crumbs protein complex, nagie oko is essential for the maintenance of epithelial cell polarity. Here, we show that nagie oko contains a predicted nuclear export and two conserved nuclear localization signals. We find that loss of the predicted nuclear export signal results in nuclear protein accumulation. We show that nagie oko nuclear import is redundantly controlled by the two nuclear localization signals and the evolutionarily conserved region 1 (ECR1), which links nagie oko with Par6-aPKC. Finally, deletion forms of nagie oko that lack nuclear import and export signals complement several nagie oko mutant defects in cell polarity and epithelial integrity. This finding provides an entry point to potentially novel and unknown roles of this important cell polarity regulator. PMID:18058913

Bit-Avragim, Nana; Rohr, Stefan; Rudolph, Franziska; Van der Ven, Peter; Fürst, Dieter; Eichhorst, Jenny; Wiesner, Burkhard; Abdelilah-Seyfried, Salim



Functional Isolation of Novel Nuclear Proteins Showing a Variety of Subnuclear Localizations  

Microsoft Academic Search

Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear

Kazuki Moriguchi; Tadzunu Suzuki; Yukihiro Ito; Yukiko Yamazaki; Yasuo Niwa; Nori Kurataa



Localization of the APECED protein in distinct nuclear structures.  


Autoimmune-polyendocrinopathy-candidiasis-ecto-dermaldystrophy (APECED) is the only systemic autoimmune disease with a monogenic background known so far revealing no association with the major histocompatibility complex region. We have recently isolated the gene defective in this syndrome and characterized several different mutations in individuals with the disorder. The novel gene, AIRE, contains a putative bipartite nuclear targeting signal predicting a nuclear location of the corresponding protein. The presence of two PHD-type zinc finger domains as well as the newly described putative DNA-binding domain, SAND, in the amino acid sequence of the APECED protein implies that it may be involved in the regulation of gene expression. Using transient expression of AIRE cDNA in mammalian cells we demonstrate here the nuclear location of the APECED protein. Immunohistochemical staining of transfected cells revealed that most of the recombinant 58 kDa APECED protein is present in the form of nuclear dots. By double immuno-fluorescence labelling we further show that these APECED-containing structures and the previously described PML nuclear bodies are largely non-overlapping. The AIRE protein was also visualized in multiple human tissues: a subset of the cells in thymus, in spleen and in lymph node showed nuclear staining with APECED antiserum. Immunofluorescence labelling of peripheral blood mononuclear leukocytes also revealed a nuclear body-like staining pattern in a fraction of these cells. These data from both in vitro and ex vivo systems, together with the predicted structural features of the APECED protein, suggest that this protein is most probably involved in the regulation of gene expression. PMID:9931333

Björses, P; Pelto-Huikko, M; Kaukonen, J; Aaltonen, J; Peltonen, L; Ulmanen, I



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

PubMed Central

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



Nuclear export of proteins and drug resistance in cancer.  


The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21(CIP), p27(KIP1), N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and II? and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase II?, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer. PMID:22209898

Turner, Joel G; Dawson, Jana; Sullivan, Daniel M



Nuclear localization of the PEP protein tyrosine phosphatase.  

PubMed Central

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h. Images

Flores, E; Roy, G; Patel, D; Shaw, A; Thomas, M L



Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors.  


Drosophila Tailless (Tll) is an orphan nuclear receptor involved in embryonic segmentation and neurogenesis. Although Tll exerts potent transcriptional repressive effects, the underlying molecular mechanisms have not been determined. Using the established regulation of knirps by tll as a paradigm, we report that repression of knirps by Tll involves Atrophin, which is related to vertebrate Atrophin-1 and Atrophin-2. Atrophin interacts with Tll physically and genetically, and both proteins localize to the same knirps promoter region. Because Atrophin proteins interact with additional nuclear receptors and Atrophin-2 selectively binds histone deacetylase 1/2 (HDAC1/2) through its ELM2 (EGL-27 and MTA1 homology 2)/SANT (SWI3/ADA2/N-CoR/TFIII-B) domains, our study establishes that Atrophin proteins represent a novel class of nuclear receptor corepressors. PMID:16481466

Wang, Lei; Rajan, Harini; Pitman, Jeffrey L; McKeown, Michael; Tsai, Chih-Cheng



HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype  

PubMed Central

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

Reeves, Raymond



Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance  

PubMed Central

Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the 3D structures under near physiological conditions, but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows specific segment(s) within a protein to be selectively examined by NMR thus significantly reducing the spectral complexity for large proteins and allowing a variety of solution-based NMR strategies to be applied. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50 kDa protein Csk (C-terminal Src Kinase) using expressed protein ligation methods.

Dongsheng, Liu; Xu, Rong; Cowburn, David



Nuclear export dynamics of RNA-protein complexes  

PubMed Central

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 of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work.

Grunwald, David; Singer, Robert H.; Rout, Michael



Nuclear export dynamics of RNA-protein complexes.  


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 of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work. PMID:21776079

Grünwald, David; Singer, Robert H; Rout, Michael



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.

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.



Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2.  


In order to re-examine the sub-cellular location of the three influenza A/NT/60/68 polymerase proteins PB1, PB2 and PA in infected cells, specific antisera for each polymerase component have been prepared by immunizing rabbits with polymerase-beta-galactosidase fusion proteins synthesized in Escherichia coli. We show that polymerase PB1, PB2, and PA are predominantly associated with the nucleus of influenza-infected MDCK cells by immunocytochemical techniques. In the case of polymerase PB2 we investigate the possibility that nuclear accumulation is an intrinsic property of the PB2 protein. Using a vaccinia-PB2 recombinant virus, we show that PB2 accumulates intra-nuclearly in monkey CV-1 cells in the absence of any other influenza protein, suggesting it contains an intrinsic nuclear signal. PMID:3023071

Jones, I M; Reay, P A; Philpott, K L



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



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)



A nuclear localization signal binding protein in the nucleolus  

PubMed Central

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.



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

PubMed Central

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

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



Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster.  

PubMed Central

Pre-mRNAs cotranscriptionally associate with a small group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. We have previously identified two genes in Drosophila melanogaster, Hrb98DE and Hrb87F (i.e., genes at 98DE and 87F encoding putative hnRNA binding proteins), which encode five protein species homologous to the mammalian A-B hnRNP proteins. The studies presented herein show that antibodies against the RNP domains of Hrb98DE reacted with 10 to 15 distinct spots of 38 to 40 kDa in the basic region of two-dimensional gels. These nuclear proteins bound single-stranded nucleic acids and were extracted from Drosophila tissue culture cells as 40 to 80S hnRNP complexes in association with 300 to 800 nucleotide fragments of RNA. The peak of poly(A)+ RNA sequences was coincident with the peak of HRB proteins in sucrose gradients, strongly suggesting that the HRB complexes identified are Drosophila hnRNP complexes. The repertoire of HRB proteins did not change significantly during embryogenesis and was similar to that observed in Drosophila tissue culture cells. Analyses with peptide-specific antisera demonstrated that the major proteins in the hnRNP complex were encoded by the two genes previously identified. Although the Drosophila HRB proteins are only approximately 60% identical throughout the RNP domains to the mammalian A-B hnRNP proteins, features of the basic pre-mRNA packaging mechanism appear to be highly conserved between D. melanogaster and mammals. Images

Raychaudhuri, G; Haynes, S R; Beyer, A L



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.



Chromatin condensation modulates access and binding of nuclear proteins  

PubMed Central

The condensation level of chromatin is controlled by epigenetic modifications and associated regulatory factors and changes throughout differentiation and cell cycle progression. To test whether changes of chromatin condensation levels per se affect access and binding of proteins, we used a hypertonic cell treatment. This shift to hyperosmolar medium increased nuclear calcium concentrations and induced a reversible chromatin condensation comparable to the levels in mitosis. However, this condensation was independent of mitotic histone H3 serine 10 phosphorylation. Photobleaching and photoactivation experiments with chromatin proteins—histone H2B-GFP and GFP-HP1?—before and after induced chromatin condensation demonstrated that hypercondensation reduced their dissociation rate and stabilized their chromatin binding. Finally, measuring the distribution of nucleoplasmic proteins in the size range from 30 to 230 kDa, we found that even relatively small proteins like GFP were excluded from highly condensed chromatin in living cells. These results suggest that structural changes in condensed chromatin by themselves affect chromatin access and binding of chromatin proteins independent of regulatory histone modifications.—Martin, R. M., Cardoso, M. C. Chromatin condensation modulates access and binding of nuclear proteins.

Martin, Robert M.; Cardoso, M. Cristina



Whole-genome screening identifies proteins localized to distinct nuclear bodies.  


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

Fong, Ka-Wing; Li, Yujing; Wang, Wenqi; Ma, Wenbin; Li, Kunpeng; Qi, Robert Z; Liu, Dan; Songyang, Zhou; Chen, Junjie



Nuclear magnetic resonance analysis of protein-DNA interactions  

PubMed Central

Recent methodological and instrumental advances in solution-state nuclear magnetic resonance have opened up the way to investigating challenging problems in structural biology such as large macromolecular complexes. This review focuses on the experimental strategies currently employed to solve structures of protein–DNA complexes and to analyse their dynamics. It highlights how these approaches can help in understanding detailed molecular mechanisms of target recognition.

Campagne, S.; Gervais, V.; Milon, A.



Properties of protein kinase C associated with nuclear membranes.  

PubMed Central

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

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



Further evidence that sperm nuclear proteins are necessary for embryogenesis.  


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

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



Ran-Binding Protein 3 Is a Cofactor for Crm1-Mediated Nuclear Protein Export  

Microsoft Academic Search

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This com- plex translocates through the nuclear pores and dissoci- ates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action

Mark E. Lindsay; James M. Holaska; Katie Welch; Bryce M. Paschal; Ian G. Macara



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


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

Qian, X; Costa, R H



Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.  


Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex. PMID:19297527

Shen, Shu; Tobery, Cynthia E; Rose, Mark D



Distinct roles for key karyogamy proteins during yeast nuclear fusion.  


During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion. PMID:19570912

Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D



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


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



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.

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.



Nuclear Actin-Related Proteins in Epigenetic Control  

PubMed Central

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

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



Fractionation of Nuclei and Analysis of Nuclear Proteins of Rat Liver and Morris Hepatoma 7777 x  

Microsoft Academic Search

SUMMARY The contributions of nuclear populations to the total profile of nuclear proteins in a tissue were examined in normal rat liver and Morris hepatoma 7777. Comparison by sodium dodecyl sulfate polyacrylamide gel electrophoresis of phenol-soluble nuclear proteins from tumor and control liver revealed additional proteins of molecular weight 60,000, 100,000, and 135,000 and the loss of proteins of about

Brian Wilson; Michael A. Lea; Giorgio Vidali


Importin -depending Nuclear Import Pathways: Role of the Adapter Proteins in the Docking and Releasing Steps  

Microsoft Academic Search

Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin . However, due to the presence of different import signals, the adapter protein of the imported molecules and importin is different for each pathway. Although the adapter for cNLS-protein is importin , the adapter for U1 snRNP is snurportin1

Christiane Rollenhagen; Petra Muhlhausser; Ulrike Kutay; Nelly Pante ´



Structural investigations of chromatin core protein by nuclear magnetic resonance.  


A complex derived from chromatin containing one molecule of each of histones H2A, H2B, H3, and H4, termed core protein, was studied by 13C and 1H nuclear magnetic resonance. 13C line widths, when analyzed and compared with those of native and thermally unfolded representative globular proteins, showed that regions of the core protein possess considerable mobility. Studies of Calpha and Cbeta line widths, and Calpha spin-spin relaxation times, show that this mobility arises from sections of random-coil polypeptide. It is argued that these regions are N-terminal "tails", attached to C-terminal globular polypeptides. The 270-MHz 1H nuclear magnetic resonance spectrum shows numerous ring current shifted resonances, indicating that the C-terminal globular domain has a precise tertiary structure. The globular domain most likely forms the histone "core" of the chromatin monomer particle, whilst the basic tails probably wind around the grooves of the double helix, enabling the basic side chains to interact with the DNA phosphate groups. Some biological implications of this model are considered. PMID:560200

Lilley, D M; Pardon, J F; Richards, B M



Nuclear import of LASP-1 is regulated by phosphorylation and dynamic protein-protein interactions.  


LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression. PMID:22665060

Mihlan, S; Reiß, C; Thalheimer, P; Herterich, S; Gaetzner, S; Kremerskothen, J; Pavenstädt, H J; Lewandrowski, U; Sickmann, A; Butt, E



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))



Transportin-SR2 mediates nuclear import of phosphorylated SR proteins  

PubMed Central

Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin ? family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains.

Lai, Ming-Chih; Lin, Ru-Inn; Tarn, Woan-Yuh



Relation Between Proteins Tertiary Structure, Tryptophan Fluorescence Lifetimes and Tryptophan S o ? 1 L b and S o ? 1 L a Transitions. Studies on ? 1 -acid Glycoprotein and ?-lactoglobulin  

Microsoft Academic Search

We measured fluorescence lifetimes and fluorescence spectra (excitation and emission) of tryptophan residues of ?1-acid glycoprotein (three Trp residues) and ?-lactoglobulin (two Trp residues) in absence and presence of 450 ?M progesterone.\\u000a Progesterone binds only to ?1-acid glycoprotein. In absence of progesterone, each of the two proteins displays three fluorescence lifetimes. Addition of\\u000a progesterone induces a partial inhibition of the S

Jihad René Albani



Reconstitution of nuclear protein transport with semi-intact yeast cells  

PubMed Central

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



Nuclear protein dysregulation in lymphoplasmacytic lymphoma/waldenstrom macroglobulinemia.  


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

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



Nuclear genes encoding plastid proteins expressed early in chloroplast development  

SciTech Connect

The overall objective of this grant was to characterize events which occur early in chloroplast biogenesis and to isolate nuclear genes encoding plastid proteins which are expressed during this developmental phase. In addition, the possible requirement of plastid transcription for the expression of the nuclear genes such as rbcS and cab was to be tested. The impetus for this research came from studies of chloroplast biogenesis in barley. We found that plastid DNA copy number was relatively high (120 copies/plastid vs 200 at maximal accumulation) in the meristematic region of the leaf base whereas plastid transcription activity was low in this plastid population. Later in chloroplast development transcription activity increased at least 5 fold per plastid or per template indicating that activation of plastid transcription occurred after most of the build up in plastid DNA per plastid. This suggested that activation of plastid DNA synthesis occurred early in chloroplast development and that nuclear genes involved in this activity must be regulated differently from genes such rbcS or cab which are expressed later in development. 3 refs., 7 figs.

Mullet, J.E.



A novel nuclear zinc finger protein EZI enhances nuclear retention and transactivation of STAT3  

PubMed Central

A novel cDNA EZI isolated as an oncostatin M- inducible gene encoded a protein containing 12 C2H2-type zinc fingers. EZI was found to transactivate the promoters that are also responsive to STAT3 and activated the acute phase response element (APRE) synergistically with STAT3. Co-immunoprecipitation demonstrated the association of EZI with STAT3, which was mediated by the N-terminal region (1–183) of EZI. The EZI mutant lacking this region showed reduced transcriptional activity, indicating that EZI and STAT3 function cooperatively through physical interaction. While EZI predominantly localized in the nucleus and enhanced the nuclear localization of STAT3, the EZI mutant lacking 11 zinc finger motifs failed to translocate into the nucleus and also inhibited nuclear localization of STAT3 as well as STAT3-mediated transactivation. These results indicate that EZI is a novel nuclear zinc finger protein that augments STAT3 activity by keeping it in the nucleus.

Nakayama, Koh; Kim, Kyung-Woon; Miyajima, Atsushi



Transcriptional repression and cell death induced by nuclear aggregates of non-polyglutamine protein  

Microsoft Academic Search

Nuclear aggregates of polyglutamine (polyQ)-expanded proteins are associated with a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxias (SCAs). The nuclear deposition of polyQ proteins correlates with rearrangements of nuclear matrix, transcriptional dysregulation, and cell death. To explore the requirement for polyQ tracks in educing such cellular responses, we examined whether a non-polyQ protein can deposit as

Lianwu Fu; Ya-sheng Gao; Elizabeth Sztul



Silencing of Nuclear Mitotic Apparatus protein (NuMA) accelerates the apoptotic disintegration of the nucleus  

Microsoft Academic Search

One main feature of apoptosis is the sequential degradation of the nuclear structure, including the fragmentation of chromatin\\u000a and caspase-mediated cleavage of various nuclear proteins. Among these proteins is the Nuclear Mitotic Apparatus protein (NuMA)\\u000a which plays a specific role in the organization of the mitotic spindle. The exact function of NuMA in the interphase nucleus\\u000a is unknown, but a

Pekka Taimen; Markku Kallajoki



The hnRNP C proteins contain a nuclear retention sequence that can override nuclear export signals  

PubMed Central

Nascent pre-mRNAs associate with the abundant heterogeneous nuclear RNP (hnRNP) proteins and remain associated with them throughout the time they are in the nucleus. The hnRNP proteins can be divided into two groups according to their nucleocytoplasmic transport properties. One group is completely restricted to the nucleus in interphase cells, whereas the other group, although primarily nuclear at steady state, shuttles between the nucleus and the cytoplasm. Nuclear export of the shuttling hnRNP proteins is mediated by nuclear export signals (NESs). Mounting evidence indicates that NES-bearing hnRNP proteins are mediators of mRNA export. The hnRNP C proteins are representative of the nonshuttling group of hnRNP proteins. Here we show that hnRNP C proteins are restricted to the nucleus not because they lack an NES, but because they bear a nuclear retention sequence (NRS) that is capable of overriding NESs. The NRS comprises approximately 78 amino acids and is largely within the auxiliary domain of hnRNP C1. We suggest that the removal of NRS-containing hnRNP proteins from pre- mRNA/mRNA is required for mRNA export from the nucleus and is an essential step in the pathway of gene expression.



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.

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.



The P1 family: a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.  

PubMed Central

Monospecific antibodies against an oligopeptide, conserved among the Mcm class of yeast replication proteins, were used to screen a human cDNA library. Eight of the isolated cDNA clones have the potential to code for sections of proteins with high sequence similarities to the yeast proteins Mcm3 and Cdc46 from Saccharomyces cerevisiae and Cdc21 from S. pombe. Our results establish a novel and highly conserved family of nuclear proteins in mammalian cells. Images

Hu, B; Burkhart, R; Schulte, D; Musahl, C; Knippers, R



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



A Bayesian network model of proteins' association with promyelocytic leukemia (PML) nuclear bodies.  


The modularity that nuclear organization brings has the potential to explain the function of aggregates of proteins and RNA. Promyelocytic leukemia nuclear bodies are implicated in important regulatory processes. To understand the complement of proteins associated with these intra-nuclear bodies, we construct a Bayesian network model that integrates sequence and protein-protein interaction data. The model predicts association with promyelocytic leukemia nuclear bodies accurately when interaction data is available. At a false positive rate of 10%, the true positive rate is almost 50%, indicated by an independent nuclear proteome reference set. The model provides strong support for further expanding the protein complement with several important regulators and a richer functional repertoire. Using special support vector machine (SVM)-nodes (equipped with string kernels), the Bayesian network is also able to produce predictions on the basis of sequence only, with an accuracy superior to that of baseline models. Supplementary Material is available online at PMID:20426694

Bodén, Mikael; Dellaire, Graham; Burrage, Kevin; Bailey, Timothy L



The Ty1 integrase protein can exploit the classical nuclear protein import machinery for entry into the nucleus  

Microsoft Academic Search

Like its retroviral relatives, the long terminal repeat retrotransposon Ty1 in the yeast Saccharomyces cerevisiae must traverse a permanently intact nuclear membrane for successful transposition and replication. For retrotransposition to occur, at least a subset of Ty1 proteins, including the Ty1 integrase, must enter the nucleus. Nuclear localiza- tion of integrase is dependent upon a C-terminal nuclear targeting sequence. However,

Laura M. McLane; Kanika F. Pulliam; Scott E. Devine; Anita H. Corbett



The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis  

PubMed Central

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.

Smoyer, Christine J.; McCroskey, Scott; Miller, Brandon D.; Weaver, Kyle J.; Delventhal, Kym M.; Unruh, Jay; Slaughter, Brian D.; Jaspersen, Sue L.



Nuclear localization of Sindbis virus nonstructural protein nsP2  

Microsoft Academic Search

In early infection, approximately 10% of nonstructural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear nsP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection.

Xiaozhong Wang; Mingxiao Ding



Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins  

SciTech Connect

A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced. The sequence predicts a protein of 261 amino acids (M/sub r/ 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids. The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA. Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as of the cDNA insert. Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation. Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA. These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms. A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells.

Almendral, J.M.; Huebsch, D.; Blundell, P.A.; Macdonald-Bravo, H.; Bravo, R.



2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins 1  

PubMed Central

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [?-32P]adenosine 5?-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F1 is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F1 but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins. ImagesFig. 5

Murray, Michael G.; Key, Joe L.



Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection.  


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 B(2) 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, B(1), and B(2). 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, E S; O'Hare, P



Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2  

PubMed Central

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.



Lamin A\\/C Binding Protein LAP2alpha Is Required for Nuclear Anchorage of Retinoblastoma Protein  

Microsoft Academic Search

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essen- tial for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathione

Ewa Markiewicz; Thomas Dechat; Roland Foisner; Christopher J. Hutchison



trnp: A conserved mammalian gene encoding a nuclear protein that accelerates cell-cycle progression.  


We herein describe a novel protein encoded by a single exon in a single-copy conserved mammalian gene. This protein, termed TMF regulated nuclear protein (TRNP), was identified in a yeast "two-hybrid" screen in which the "BC box" containing protein-TMF/ARA160 served as a bait. TRNP is a basic protein which accumulates in an insoluble nuclear fraction in mammalian cells. It is 227 aa long in humans and chimps and 223 aa long in mice. Enforced expression of TRNP in cells that do not express this protein significantly increased their proliferation rate by enhancing their cell-cycle progression from the G0/G1 to the S phase. Like another proliferation promoting factor, Stat3, TRNP was directed to proteasomal degradation by TMF/ ARA160. Thus, the trnp gene encodes a novel mammalian conserved nuclear protein that can accelerate cellcycle progression and is regulated by TMF/ARA160. PMID:16792503

Volpe, Marina; Shpungin, Sally; Barbi, Chany; Abrham, Galya; Malovani, Hanna; Wides, Ron; Nir, Uri



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.



Nuclear Receptor Interaction Protein (NRIP) expression assay using human tissue microarray and immunohistochemistry technology confirming nuclear localization  

Microsoft Academic Search

Background  A novel human nuclear receptor interaction protein (NRIP) has recently been discovered by Chen SL et al, which may play a\\u000a role in enhancing the transcriptional activity of steroid nuclear receptors in prostate (LNCaP) and cervical (C33A) cancer\\u000a cell lines. However, knowledge about the biological functions and clinical implications of NRIP, is still incomplete. Our\\u000a aim was to determine the

Chih-Ping Han; Ming-Yung Lee; Shu-Ling Tzeng; Chung-Chin Yao; Po-Hui Wang; Ya-Wen Cheng; Show-Li Chen; Teresa S Wu; Yeu-Sheng Tyan; Lai-Fong Kok



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:



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

PubMed Central

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

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




PubMed Central

Integral proteins of the nuclear envelope inner membrane have been proposed to reach their sites by diffusion after their co-translational insertion in the rough endoplasmic reticulum. They are then retained in the inner nuclear membrane by binding to nuclear structures. One such structure is the nuclear lamina, an intermediate filament meshwork composed of A-type and B-type lamin proteins. Emerin, MAN1 and LBR are three integral inner nuclear membrane proteins. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts from wild-type mice and Lmna -/- mice, which lack A-type lamins. We then studied the diffusional mobilities of emerin, MAN1 and LBR using fluorescence recovery after photobleaching. We show that emerin and MAN1, but not LBR, are more mobile in the inner nuclear membrane of cells from Lmna -/- mice than in cells from wild-type mice. In cells from Lmna -/- mice expressing exogenous lamin A, the protein mobilities were similar to those in cells from wild-type mice. This supports a model where emerin and MAN1 are at least partly retained in the inner nuclear membrane by binding to A-type lamins, while LBR depends on other binding partners for its retention.

Ostlund, Cecilia; Sullivan, Teresa; Stewart, Colin L.; Worman, Howard J.



The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor.  


The androgen receptor (AR) is a transcription factor that mediates androgen action. We have used the green fluorescent protein (GFP) technique to investigate dynamics of nuclear trafficking of human AR in living cells. In the absence of ligand, the GFP-AR fusion protein is distributed between cytoplasm and nuclei. Androgen exposure leads to a rapid and complete import of GFP-AR to nuclei of CV-1 cells (>=90% nuclear in 30 minutes), whereas a pure antiandrogen, Casodex, elicits a slower (<40% nuclear in 30 minutes) and incomplete transfer. Unliganded ARs with mutations in the basic amino acids of the bipartite nuclear localization signal (NLS) within the second zinc finger and the hinge region are predominantly cytoplasmic and their androgen-dependent nuclear import is severely compromised ((3/4)20% nuclear in 30 minutes). Interestingly, substitutions of the Leu residues flanking the bipartite NLS lead to inefficient nuclear transfer in response to androgen ((3/4)20% nuclear in 30 minutes). The ligand-binding domain of AR, which represses bipartite NLS activity, contains an agonist-specific NLS. The small nuclear RING finger protein SNURF, which interacts with AR through a region overlapping with the bipartite NLS, facilitates AR import to nuclei and retards its export on hormone withdrawal. More AR is associated with the nuclear matrix in the presence than absence of coexpressed SNURF. We suggest that the SNURF-mediated tethering of AR in nuclei represents a novel mechanism for activating steroid receptor functions. PMID:10934038

Poukka, H; Karvonen, U; Yoshikawa, N; Tanaka, H; Palvimo, J J; Jänne, O A



[Changes of nuclear matrix proteins during differentiation of human osteosarcoma MG-63 cells induced by HMBA].  


Human osteosarcoma MG-63 cells were induced into differentiation by hexamethylene bisacetamide (HMBA). Its nuclear matrix proteins were selectively extracted, and subjected to two dimensional gel electrophoresis analysis. The resulted protein patterns were analyzed by Melanie software. There were 12 spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The up-regulated proteins were identified as MHC class II antigen, interferon-stimulated gene factor 3d, hypothetical protein DKFZp434M2221.1,8-hydroxy-guanine glycosylase homolog ogg1, and vimentin. The down-regulated ones were hnRNP A2/B1 and actin; and two newly expressed proteins under induction were 60S ribosomal protein L21 and ST2 protein. This study suggests that the induced differentiation of carcinoma cells is accompanied with changes of nuclear matrix proteins, and confirms the presence of some specific nuclear matrix proteins associated with carcinoma cell growth and differentiation. The changed nuclear matrix proteins are potential markers for cancer diagnosis or targets for cancer therapy. PMID:16944589

Zhao, Chun Hong; Li, Qi Fu; Li, Zhi Xing; Chen, Jin An



The Protein Encoded by Oncogene 6b from Agrobacterium tumefaciens Interacts with a Nuclear Protein of Tobacco  

PubMed Central

The 6b gene in the T-DNA from Agrobacterium has oncogenic activity in plant cells, inducing tumor formation, the phytohormone-independent division of cells, and alterations in leaf morphology. The product of the 6b gene appears to promote some aspects of the proliferation of plant cells, but the molecular mechanism of its action remains unknown. We report here that the 6b protein associates with a nuclear protein in tobacco that we have designated NtSIP1 (for Nicotiana tabacum 6b–interacting protein 1). NtSIP1 appears to be a transcription factor because its predicted amino acid sequence includes two regions that resemble a nuclear localization signal and a putative DNA binding motif, which is similar in terms of amino acid sequence to the triple helix motif of rice transcription factor GT-2. Expression in tobacco cells of a fusion protein composed of the DNA binding domain of the yeast GAL4 protein and the 6b protein activated the transcription of a reporter gene that was under the control of a chimeric promoter that included the GAL4 upstream activating sequence and the 35S minimal promoter of Cauliflower mosaic virus. Furthermore, nuclear localization of green fluorescent protein–fused 6b protein was enhanced by NtSIP1. A cluster of acidic residues in the 6b protein appeared to be essential for nuclear localization and for transactivation as well as for the hormone-independent growth of tobacco cells. Thus, it seems possible that the 6b protein might function in the proliferation of plant cells, at least in part, through an association with NtSIP1.

Kitakura, Saeko; Fujita, Tomomichi; Ueno, Yoshihisa; Terakura, Shinji; Wabiko, Hiroetsu; Machida, Yasunori



Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis  

PubMed Central

Background The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation. Results Using transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling. Conclusion This work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies.

Chen, I-Hsiung Brandon; Huber, Michael; Guan, Tinglu; Bubeck, Anja; Gerace, Larry



An improved high throughput protein-protein interaction assay for nuclear hormone receptors  

PubMed Central

The Glutathione-S-Transferase (GST) “pulldown” assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay.

Goodson, Michael L.; Farboud, Behnom; Privalsky, Martin L.



Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy  

Microsoft Academic Search

The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ?30 different proteins, termed nucleoporins, whose arrangement within the NPC is an important unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We

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



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:



Evolving Regular Expression-Based Sequence Classifiers for Protein Nuclear Localisation  

Microsoft Academic Search

A number of bioinformatics tools use regular expression (RE) matching to locate protein or DNA sequence motifs that have been discovered by researchers in the laboratory. For exam- ple, patterns representing nuclear localisation signals (NLSs) are used to predict nuclear lo- calisation. NLSs are not yet well understood, and so the set of currently known NLSs may be incomplete. Here

Amine Heddad; Markus Brameier; Robert M. Maccallum



On the origin of the polyhedral protein of the nuclear polyhedrosis virus of Autographa californica  

Microsoft Academic Search

The purpose of this study was to investigate the origin of the polyhedral protein of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), one of the best characterized viruses of the family Baculoviridae. The present knowledge of the properties of the nuclear polyhedrosis virus of Autographa californica, are described in chapter 1.One of the most striking features

Beek van der C. P



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



A nuclear export signal within the structural Gag protein is required for prototype foamy virus replication  

Microsoft Academic Search

BACKGROUND: The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV), Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as

Noémie Renault; Joelle Tobaly-Tapiero; Joris Paris; Marie-Lou Giron; Audrey Coiffic; Philippe Roingeard; Ali Saïb



Nuclear Matrix Protein Alterations Associated with Colon Cancer and Colon Metastasis to the Liver and Use Thereof.  

National Technical Information Service (NTIS)

Proteins useful in the diagnosis of proliferative disorders of the colon are present in nuclear matrix protein preparations and can be characterized by molecular weight, isoelectric point, and amino acid sequence. The proteins may be identified, for examp...

A. J. Bauer G. Bruenagel R. H. Getzenberg R. E. Schoen



Isolation of nuclear proteins from flax (Linum usitatissimum L.) seed coats for gene expression regulation studies  

PubMed Central

Background While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds. Findings In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments. Conclusions Routinely, around 250 ?g of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies.



Characterization of cardiac muscle factor 1 sequence motifs: retinoblastoma protein binding and nuclear localization.  


Cardiac muscle factor 1 (CMF1) is characterized as a protein important in cardiac and skeletal myocyte differentiation and is expressed in a developmentally regulated manner. Sequence analysis data showed that CMF1 has crucial protein-protein interaction domains, has a retinoblastoma protein-binding site which interacts with RB directly in vitro and in the embryo, has a functional nuclear localization signal and is highly homologous to other cell cycle regulatory proteins such as mitosin and centromere protein F, which suggests that CMF1 may be involved in regulating the cell cycle. PMID:11814677

Redkar, Abhay; deRiel, Jon K; Xu, Yong-Sheng; Montgomery, Michael; Patwardhan, Vidya; Litvin, Judith



Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro.  


Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, 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 transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components. PMID:15123622

Citovsky, Vitaly; Kapelnikov, Anat; Oliel, Shachar; Zakai, Nehama; Rojas, Maria R; Gilbertson, Robert L; Tzfira, Tzvi; Loyter, Abraham



A novel splicing regulator shares a nuclear import pathway with SR proteins.  


Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin beta-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus. PMID:12628928

Lai, Ming-Chih; Kuo, Hao-Wei; Chang, Wen-Cheng; Tarn, Woan-Yuh



A novel splicing regulator shares a nuclear import pathway with SR proteins  

PubMed Central

Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin ?-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.

Lai, Ming-Chih; Kuo, Hao-Wei; Chang, Wen-Cheng; Tarn, Woan-Yuh



Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin  

PubMed Central

Background Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1? (HP1?) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D. Results We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1?, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1?, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1? or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. Conclusion Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.



Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.  


Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface. PMID:23181366

Buanne, Pasquale; Renzone, Giovanni; Monteleone, Francesca; Vitale, Monica; Monti, Simona Maria; Sandomenico, AnnaMaria; Garbi, Corrado; Montanaro, Donatella; Accardo, Marina; Troncone, Giancarlo; Zatovicova, Miriam; Csaderova, Lucia; Supuran, Claudiu T; Pastorekova, Silvia; Scaloni, Andrea; De Simone, Giuseppina; Zambrano, Nicola



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

PubMed Central

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

Mao, Grace; Brody, James P.



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

SciTech Connect

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

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



The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins  

PubMed Central

The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo- cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin ?/?-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.

Walther, Tobias C.; Fornerod, Maarten; Pickersgill, Helen; Goldberg, Martin; Allen, Terry D.; Mattaj, Iain W.



Intranuclear filaments containing a nuclear pore complex protein  

Microsoft Academic Search

Nuclear pore complexes (NPCs) are an- choring sites of intranuclear filaments of 3-6 nm di- ameter that are coaxially arranged on the perimeter of a cylinder and project into the nuclear interior for lengths varying in different kinds of cells. Using a specific monoclonal antibody we have found that a polypeptide of ~190 kD on SDS-PAGE, which ap- pears to

Volker C. Cordes; Sonja Reidenbach; Andreas Kiihler; Nico Stuurman; Roel van Driel; Werner W. Franke



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

PubMed Central

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation 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 that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently cloned FANCG/XRCC9 protein is required for binding of the FANCA and FANCC proteins. Moreover, the FANCG protein is a component of a nuclear protein complex containing FANCA and FANCC. The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex. Our results demonstrate that the three cloned FA proteins cooperate in a large multisubunit complex. Disruption of this complex results in the specific cellular and clinical phenotype common to most FA complementation groups.

Garcia-Higuera, Irene; Kuang, Yanan; Naf, Dieter; Wasik, Jennifer; D'Andrea, Alan D.



Prolactin-provoked alterations of cytosolic, membrane, and nuclear protein kinase C following partial hepatectomy  

Microsoft Academic Search

The adenohypophyseal polypeptide hormone prolactin is a potent liver mitogen, stimulating cell cycle progression, an effect that appears coupled to increasing protein kinase C activity in membrane and nuclear fractions. Here, we examine whether hepatocyte proliferation, stimulated by partial hepatectomy, is associated with altered serum prolactin or protein kinase C activation. Within 5–15 min of liver resection, serum prolactin concentrations

Arthur R. Buckley; Paul D. Crowe; Patricia A. Bauman; Leigh A. Neumayer; Hugh E. Laird; Diane Haddock Russell; Charles W. Putnam



PML bodies control the nuclear dynamics and function of the CHFR mitotic checkpoint protein  

Microsoft Academic Search

Nuclear foci containing the promyelocytic leukemia protein (PML bodies), which occur in most cells, play a role in tumor suppression. Here, we demonstrate that CHFR, a mitotic checkpoint protein frequently inactivated in human cancers, is a dynamic component of PML bodies. Intermolecular fluorescence resonance energy transfer analysis identified a distinct fraction of CHFR that interacts with PML in living cells.

Matthew J Daniels; Alexander Marson; Ashok R Venkitaraman



Nuclear localization is required for function of the essential clock protein FRQ.  

PubMed Central

The frequency (frq) gene in Neurospora encodes central components of a circadian oscillator, a negative feedback loop involving frq mRNA and two forms of FRQ protein. Here we report that FRQ is a nuclear protein and nuclear localization is essential for its function. Deletion of the nuclear localization signal (NLS) renders FRQ unable to enter into the nucleus and abolishes overt circadian rhythmicity, while reinsertion of the NLS at a novel site near the N-terminus of FRQ restores its function. Each form of FRQ enters the nucleus soon after its synthesis in the early subjective day; there is no evidence for regulated sequestration in the cytoplasm prior to nuclear entry. The kinetics of the nuclear entry are consistent with previous data showing rapid depression of frq transcript levels following the synthesis of FRQ, and suggest that early in each circadian cycle, when FRQ is synthesized, it enters the nucleus and depresses the level of its own transcript.

Luo, C; Loros, J J; Dunlap, J C



Conformations and conformational dynamics of proteins in solution studied by nuclear magnetic double resonance  

SciTech Connect

Saturation of single resonances in the proton nuclear magnetic resonance (NMR) spectrum of a protein in solution results in changes in intensities of other resonances. These changes come about either through nuclear Overhauser effects which arise because of dipolar coupling between the protons in the molecule, or through cross-saturation effects which arise when the protein fluctuates between different well defined conformations. For lysozyme from hen egg white (mol. wt. approx. 14,500) the method reveals details of the protein structure and of individual atom fluctuations.

Dobson, C.M.; Hoch, J.C.; Olejniczak, E.T.; Poulsen, F.M.



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.

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



RNA recognition motifs involved in nuclear import of RNA-binding proteins.  


In eukaryotic cells, a regulated import and export of factors is required to fulfill the requirements of precise gene expression. Post-transcriptional regulation of gene expression has proven to provide ubiquitous control, as well as a quick response to environmental changes when required. RNA-binding proteins (RBP) are involved in the several steps at which mRNA biogenesis, stability, translation and decay is exerted. The most characterized RBPs contain single or multiple copies of an RNA Recognition Motif (RRM). Here, we concentrate on RRMs mediating protein nuclear import by virtue of its ability to interact with proteins, besides interacting with nucleic acids. The consensus on how RRM-protein interactions take place is non-existent, and so is the involvement of the RRM as a nuclear localization signal (NLS). Within the cases examined, the single RRM from a trypanosome RBP behaves as a structural NLS, alternating nuclear import and RNA-binding. PMID:20458169

Cassola, Alejandro; Noé, Griselda; Frasch, Alberto C



Mitochondrial malate dehydrogenases in Brassica napus: altered protein patterns in different nuclear mitochondrial combinations  

Microsoft Academic Search

Two-dimensional analyses of mitochondrial proteins of Brassica napus revealed a set of differences in patterns of mitochondrial matrix proteins isolated from different nuclear backgrounds. One of these varying proteins was identified as mitochondrial malate dehydrogenase (mMDH; EC by homology analyses of the partial amino acid sequence. Immunological detection identified additional mMDH subunits and detected different patterns of mMDH subunits

Uwe Witt; Renate Lührs; Friedrich Buck; Kerstin Lembke; Marlies Grüneberg-Seiler; Wolfgang Abel



Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form  

Microsoft Academic Search

THE Ran proteins constitute a distinct branch of the superfamily of Ras-related GTP-binding proteins1 which function as molecular switches cycling between GTP-bound 'on' and GDP-bound 'off states2. Ran is located predominantly in the nucleus of eukaryotic cells3 and is involved in the nuclear import of proteins4,5 as well as in control of DNA synthesis and of cell-cycle progression6-8. We report

Klaus Scheffzek; Christian Klebe; Karin Fritz-Wolf; Wolfgang Kabsch; Alfred Wittinghofer



Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins  

Microsoft Academic Search

The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa ß subunits of F1-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial

James Whelan; Carina Knorpp; Matthew A. Harmey; Elzbieta Glaser



KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development.  


Nuclear movement relative to cell bodies is a fundamental process during certain aspects of mammalian retinal development. During the generation of photoreceptor cells in the cell division cycle, the nuclei of progenitors oscillate between the apical and basal surfaces of the neuroblastic layer (NBL). This process is termed interkinetic nuclear migration (INM). Furthermore, newly formed photoreceptor cells migrate and form the outer nuclear layer (ONL). In the current study, we demonstrated that a KASH domain-containing protein, Syne-2/Nesprin-2, as well as SUN domain-containing proteins, SUN1 and SUN2, play critical roles during INM and photoreceptor cell migration in the mouse retina. A deletion mutation of Syne-2/Nesprin-2 or double mutations of Sun1 and Sun2 caused severe reduction of the thickness of the ONL, mislocalization of photoreceptor nuclei and profound electrophysiological dysfunction of the retina characterized by a reduction of a- and b-wave amplitudes. We also provide evidence that Syne-2/Nesprin-2 forms complexes with either SUN1 or SUN2 at the nuclear envelope to connect the nucleus with dynein/dynactin and kinesin molecular motors during the nuclear migrations in the retina. These key retinal developmental signaling results will advance our understanding of the mechanism of nuclear migration in the mammalian retina. PMID:21177258

Yu, Juehua; Lei, Kai; Zhou, Min; Craft, Cheryl M; Xu, Gezhi; Xu, Tian; Zhuang, Yuan; Xu, Rener; Han, Min



Selective displacement of nuclear proteins by antitumor drugs having affinity for nucleic acids.  


The nuclear chromatin binding sites of the antitumor drugs mitoxantrone, ametantrone, doxorubicin, mithramycin, and actinomycin D and the intercalating ligand ethidium were studied by polyacrylamide gel electrophoresis of the proteins released from rat liver nuclei in the presence and absence of these drugs in buffer of low ionic strength (10 mM NaCl). At 25-50 microM free ligand concentration, each drug produced a specific and reproducible pattern of extractable proteins of different molecular weight by (i) releasing new proteins, (ii) altering the quantity of particular extracted proteins, and/or (iii) selectively entrapping other proteins in the nuclei. Ethidium, up to 100 microM, did not affect release of proteins from the nuclei. These results indicate that each ligand either has different binding site(s) in chromatin or modulates chromatin structure in a specific way by changing the affinity of different sets of proteins for their respective binding sites, resulting in their selective extraction or entrapment. The lack of effect of ethidium indicates that intercalation of the ligand to DNA, per se, does not alter the release of nuclear proteins. If patterns of nuclear proteins selectively released or retained by antitumor drugs are found to correlate with biological activity, this type of analysis may be helpful in new drug design and screening. PMID:2525781

Bartkowiak, J; Kapuscinski, J; Melamed, M R; Darzynkiewicz, Z



Intranuclear membranes induced by lipidated proteins are derived from the nuclear envelope  

PubMed Central

Association of nuclear lamins with the inner nuclear membrane (INM) is mediated by lipid modifications: either by C-terminal isoprenylation or N-terminal myristoylation. Overexpression of lamins or other lipidated nuclear proteins induces the formation of intranuclear membrane-like arrays. Lamin-induced intranuclear array formation has been observed in Xenopus oocytes as well as in mammalian tissue culture cells. With the use of a membrane-specific fluorescence dye we show here that these arrays are made up of typical lipid membranes. While continuity between these intranuclear membranes and the INM has not been observed so far the presence of integral as well as luminal marker proteins of the endoplasmic reticulum (ER) indicates that these membranes are derived from the nuclear membrane/ER compartment. Earlier studies demonstrated that overexpression of integral membrane proteins of the INM can induce formation of intranuclear membranes, which bud from the INM. Integral membrane proteins reach the INM via the pore membranes while lipidated proteins are imported into the nucleoplasm via the classical NLS pathway where they interact with the INM via their lipid moieties. Together with the previously published data our results show that the formation of intranuclear membranes follows similar routes irrespective of whether the proteins triggering membrane formation are integral membrane or lipidated proteins.

Linde, Nina



Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr  

Microsoft Academic Search

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucle- oporins, but the studies have from necessity probed binding events

Sundeep Shah; Stuart Tugendreich; Douglass Forbes



Nuclear localization and shuttling of herpes simplex virus tegument protein VP13/14.  


The herpes simplex virus type 1 gene UL47 encodes the tegument proteins referred to collectively as VP13/14, which are believed to be differentially modified forms of the same protein. Here we show that the major product of the UL47 gene during transient expression is VP14, suggesting that some feature of virus infection is required to produce VP13. We have tagged VP13/14 with green fluorescent protein and have demonstrated that the protein is targeted efficiently to the nucleus, where it often localizes in numerous punctate domains. Furthermore, we show that removal of the N-terminal 127 residues of the protein abrogates nuclear accumulation, and we have identified a 14-amino-acid peptide from this region that is sufficient to function as a nuclear targeting signal and transport a heterologous protein to the nucleus. This short peptide contains two runs of four arginine residues, suggesting that the VP13/14 nuclear localization signal may behave in a manner similar to that of the arginine-rich nuclear localization signals of the retrovirus transactivator proteins Tat, Rev, and Rex. In addition, by using heterokaryon assays, we show that VP13/14 is capable of shuttling between the nucleus and cytoplasm of the cell, a property that may be attributed to three leucine-rich stretches in the C-terminal half of the protein that again bear similarity to the nuclear export signals of Rev and Rex. This is the first demonstration of a tegument protein that is specifically targeted to the nucleus, a feature which may be relevant both during virus entry, when VP13/14 enters the cell as a component of the tegument, and at later times, when large amounts of newly synthesized VP13/14 are present within the cell. PMID:11222679

Donnelly, M; Elliott, G



The CRM1 nuclear export protein in normal development and disease  

PubMed Central

CRM1 (Chromosomal Maintenance 1, also known as Exportin 1) is the major mammalian export protein that facilitates the transport of large macromolecules including RNA and protein across the nuclear membrane to the cytoplasm. The gene encoding CRM1 was originally identified in yeast as required to maintain higher order chromosome structure. In mammalian cells, CRM1 was found to bind several nuclear pore proteins hence its role in nuclear-cytosolic transport was discovered. In addition to nuclear-cytosolic transport, CRM1 also plays a role in centrosome duplication and spindle assembly, especially in response to DNA damage. The crystal structure of CRM1 suggests a complex protein that binds the Ran protein bound to GTP, allowing for a conformational change that facilitates binding to different cargo proteins through a nuclear export signal (NES). Included in the cadre of cargo are multiple tumor suppressor and oncoproteins as p53, BRCA1, Survivin, NPM, and APC, which function in the nucleus to regulate transcription or aid in chromosomal assembly and movement. An imbalance in the cytosolic level of these proteins has been observed in cancer cells, resulting in either inactivation (tumor suppressor) or an excess of anti-apoptotic activity (oncoprotein). Thus, the concept of inhibiting CRM1 has been explored as a potential therapeutic intervention. Indeed, inhibition of CRM1 by a variety of small molecules that interfere with cargo-NES binding results in cancer cell death. Whether all of these proteins together are responsible for this phenotype or whether specific proteins are required for this effect is unclear at this time.

Nguyen, Kevin T; Holloway, Michael P; Altura, Rachel A



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


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

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



Cocksfoot mottle sobemovirus coat protein contains two nuclear localization signals  

Microsoft Academic Search

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced\\u000a green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal\\u000a amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized

Allan Olspert; Heiti Paves; Raavo Toomela; Tiina Tamm; Erkki Truve



Carboxy terminal modifications of the P0 protein reveal alternative mechanisms of nuclear ribosomal stalk assembly  

PubMed Central

The P0 scaffold protein of the ribosomal stalk is mainly incorporated into pre-ribosomes in the cytoplasm where it replaces the assembly factor Mrt4. In analyzing the role of the P0 carboxyl terminal domain (CTD) during ribosomal stalk assembly, we found that its complete removal yields a protein that is functionally similar to Mrt4, whereas a chimeric Mrt4 containing the P0 CTD behaves more like P0. Deleting the P0 binding sites for the P1 and P2 proteins provoked the nuclear accumulation of P0?AB induced by either leptomycin B-mediated blockage of nuclear export or Mrt4 deletion. This effect was reversed by removing P1/P2 from the cell, whereas nuclear accumulation was restored on reintroduction of these proteins. Together, these results indicate that the CTD determines the function of the P0 in stalk assembly. Moreover, they indicate that in cells lacking Mrt4, P0 and its stalk base partner, the L12 protein, bind to pre-ribosomes in the nucleus, a complex that is then exported to the cytoplasm by a mechanism assisted by the interaction with P1/P2 proteins. Furthermore, in wild-type cells, the presence of nuclear pre-ribosome complexes containing P0 but not L12 is compatible with the existence of an alternative stalk assembly process.

Francisco-Velilla, Rosario; Remacha, Miguel; Ballesta, Juan P.G.



0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein  

SciTech Connect

Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and round spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.

Zhang Heng; Denhard, Leslie A.; Zhou Huaxin; Liu Lanhsin [Birth Defects Center, University of Louisville Health Sciences Center, Dental Building Room 203B, 501 S. Preston Street, Louisville, KY 40202 (United States); Lan Zijian [Birth Defects Center, University of Louisville Health Sciences Center, Dental Building Room 203B, 501 S. Preston Street, Louisville, KY 40202 (United States)], E-mail:



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:; 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)



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:



Stage transitions in B-lymphocyte differentiation correlate with limited variations in nuclear proteins.  


Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages. PMID:2000390

Rabilloud, T; Pennetier, J L; Hibner, U; Vincens, P; Tarroux, P; Rougeon, F



Stage transitions in B-lymphocyte differentiation correlate with limited variations in nuclear proteins.  

PubMed Central

Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages. Images

Rabilloud, T; Pennetier, J L; Hibner, U; Vincens, P; Tarroux, P; Rougeon, F



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

Microsoft Academic Search

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

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



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

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



Dynamic correlation networks in human peroxisome proliferator-activated receptor-? nuclear receptor protein  

Microsoft Academic Search

Peroxisome proliferator-activated receptor-? nuclear receptor (PPAR-?) belongs to the superfamily of nuclear receptor proteins\\u000a that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism.\\u000a A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-?. Here\\u000a we use normal-mode analysis of PPAR-? to characterize a

Jeremy Fidelak; Silvia Ferrer; Michael Oberlin; Dino Moras; Annick Dejaegere; Roland H. Stote



Alterations in the nuclear architecture produced by the overexpression of tau protein in neuroblastoma cells.  


Abnormal intracellular aggregation of tau protein is a pathological condition leading to neuronal death in Alzheimer's disease. Fibrillar and nonfibrillar aggregates of tau protein alter the normal functioning of neurons by disturbing important cellular processes and distinct membranous organelles. However, tau-caused alterations in the nuclear compartment are not totally established so far. In our study we evaluated whether tau protein and its Asp421-truncated variant produce alterations in the normal architecture of the nucleus when expressed in cultured neuroblastoma cells. After 48 hours of transfection, significant deformity of the nuclear compartment with extensive lobulations along the nuclear envelope was observed in SH-SY5Y cells expressing either full-length tau or Asp421-truncated tau. This aberrant formation did not involve either nuclear fragmentation or cell death. The lobulated nuclei were devoid of tau protein, which mostly remained in the cytoplasm in a nonfibrillar state. Degradation of nuclear Lamins was not observed in tau-expressing SH-SY5Y cells, and a cell-cycle analysis did not show aberrant chromosome accumulation. Thus multiple division defects leading to multinucleation were discarded. The lobulated nuclei in tau-expressing SH-SY5Y cells seem to more resemble the multilobular phenotype of the nuclear envelope seen in Lamin-mutated cells from those pathological conditions leading to premature aging. Nevertheless, in our tau-expressing cells, the abnormal formation of cortical and perinuclear rings of tubulin generated by tau binding may be a more feasible mechanism of a nuclear-cytoskeleton generating force that causes the nuclear deformation. PMID:23635409

Monroy-Ramírez, Hugo C; Basurto-Islas, Gustavo; Mena, Raul; Cisneros, Bulmaro; Binder, Lester I; Avila, Jesús; Garcia-Sierra, Francisco



Nuclear localization signals in the Xenopus FGF embryonic early response 1 protein  

Microsoft Academic Search

Xenopus early response 1 (XER1) is a fibroblast growth factor-inducible transcription factor whose developmentally regulated nuclear localization is thought to be important in the control of cell differentation during embryonic development [Luchman et al., Mech. Dev. 80 (1999) 111–114]. Analysis of the XER1 amino acid sequence revealed four regions which contain potential nuclear localization sequences (NLSs). Using mutant XER1 proteins

Janine N. Post; Laura L. Gillespie; Gary D. Paterno



Interactions and three-dimensional localization of a group of nuclear pore complex proteins  

Microsoft Academic Search

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A mono- clonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cul- tured cell lines by immunofluorescence microscopy. These

N. Pante; Ricardo Bastos; Isabel McMorrow; Brian Burke; Ueli Aebi



Heat Shock Protein 90 Is an Essential Molecular Chaperone for Nuclear Transport of Glucocorticoid Receptor  

Microsoft Academic Search

PURPOSE. Glucocorticoid sensitivity in glaucoma has been attrib- uted to differences in the expression of the two glucocorticoid receptors, GR and GR .G R undergoes steroid-dependent nu- clear translocation by associating with a heat shock protein (Hsp)90 multiprotein heterocomplex. The nuclear transport of the non-ligand-binding GR is still unknown. In this study, the roles of Hsp90 in the nuclear transport

Xinyu Zhang; Abbot F. Clark; Thomas Yorio



Nuclear localization of dengue virus capsid protein is required for DAXX interaction and apoptosis  

Microsoft Academic Search

Dengue virus capsid protein (DENVC) localizes to both the cytoplasm and nucleus of dengue virus-infected cells. DENV C contains three nuclear localization signals (NLS), 6KKAR9, 73KKSK76, and the bipartite signal 85RKeigrmlnilnRRRR100. Stable HepG2 cells constitutively expressing DENV C, DENV C (?85–100) and DENV C (?73–100) were constructed to clarify whether nuclear translocation of DENV C affected apoptosis in liver cell

Janjuree Netsawang; Sansanee Noisakran; Chunya Puttikhunt; Watchara Kasinrerk; Wiyada Wongwiwat; Prida Malasit; Pa-thai Yenchitsomanus; Thawornchai Limjindaporn



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



Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry  

Microsoft Academic Search

Herewereporttherapididentificationofthe proteins of the spliceosomal U1 small nuclear ribonucleopro- tein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence da- tabases. 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.AU1snRNPfractioncontaining20proteins was obtained. Further purification by glycerol gradient cen- trifugationidentifiednineU1snRNPspecificandsixcommon proteins. The

A LEXANDERGOTTSCHALK; Emil Mannkopff-Strasse


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.

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



[Detection and isolation of glycoproteins and nuclear proteins in the bovine brain].  


Using ammonium sulphate precipitation, ion exchange chromatography and preparative isoelectrofocusing, 9 organ-specific glycoproteins and 16 specific nuclear proteins were isolated from bovine brain nervous tissue in a homogeneous state. The isolation of proteins was controlled by a solid phase immunoenzymatic analysis. The molecular weight, subunit composition and isoelectric points of the proteins were determined and their ability to interact with immobilized calf thymus DNA and concanavalin A was demonstrated. It was assumed that the multiplicity of specific proteins of brain tissue is a molecular basis which provides for the functional specificity of the nervous tissue at large. PMID:6525367

Mekhtiev, A A; Gruden', M A; Poletaev, A B



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

SciTech Connect

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 protein (YFP), with respect to these bundle arrays. At 37 deg C, the nucleus-targeted, temperature-sensitive Xenopus vimentin was deposited in focal accumulations. Upon shift to 28 deg C, polymerization was induced and filament arrays became visible. Within 2 h after temperature shift, arrays were found to be composed of filaments loosely embedded in the nucleoplasm. The filaments were restricted to limited areas of the nucleus between focal accumulations. Upon incubation at 28 deg C for several hours, NLS vimentin filaments formed bundles looping throughout the nuclei. Speckles and Cajal bodies frequently localized in direct neighborhood to vimentin bundles. Similarly, small crystalline particles formed by YFP-tagged Mx1 also located next to vimentin bundles. Taking into account that nuclear targeted vimentin locates in the interchromosomal domain (ICD), we conclude that nuclear body-sized particles share a common nuclear space which is controlled by higher order chromatin organization.

Richter, Karsten [Division Molecular Genetics, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany); Reichenzeller, Michaela [Division Biology of the Cell, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany); Goerisch, Sabine M. [Division Molecular Genetics, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany); Schmidt, Ute [Division Molecular Genetics, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany); Scheuermann, Markus O. [Division Molecular Genetics, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany); Herrmann, Harald [Division Biology of the Cell, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany); Lichter, Peter [Division Molecular Genetics, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg (Germany)]. E-mail:



How a disordered ubiquitin ligase maintains order in nuclear protein homeostasis  

PubMed Central

Cells use protein quality control (PQC) systems to protect themselves from potentially harmful misfolded proteins. Many misfolded proteins are repaired by molecular chaperones, but irreparably damaged proteins must be destroyed. Eukaryotes predominantly destroy these abnormally folded proteins through the ubiquitin-proteasome pathway, which requires compartment-specific ubiquitin ligase complexes that mark substrates with ubiquitin for proteasome degradation. In the yeast nucleus, misfolded proteins are targeted for degradation by the ubiquitin ligase San1, which binds misfolded nuclear proteins directly and does not appear to require chaperones for substrate binding. San1 is also remarkably adaptable, as it is capable of ubiquitinating a structurally diverse assortment of abnormally folded substrates. We attribute this adaptability to San1's high degree of structural disorder, which provides flexibility and allows San1 to conform to differently shaped substrates. Here we review our recent work characterizing San1's distinctive mode of substrate recognition and the associated implications for PQC in the nucleus.

Rosenbaum, Joel C



Nuclear photosynthetic gene expression is synergistically modulated by rates of protein synthesis in chloroplasts and mitochondria.  


Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5'-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression. PMID:16517761

Pesaresi, Paolo; Masiero, Simona; Eubel, Holger; Braun, Hans-Peter; Bhushan, Shashi; Glaser, Elzbieta; Salamini, Francesco; Leister, Dario



NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins.  


We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses. PMID:23500724

Papageorgiou, Dimitris N; Demmers, Jeroen; Strouboulis, John



Sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to ICP0 domains.  


The subcellular localization of herpes simplex virus tegument proteins during infection is varied and complex. By using viruses expressing tegument proteins tagged with fluorescent proteins, we previously demonstrated that the major tegument protein VP22 exhibits a cytoplasmic localization, whereas the major tegument protein VP13/14 localizes to nuclear replication compartments and punctate domains. Here, we demonstrate the presence of a second minor population of VP22 in nuclear dots similar in appearance to those formed by VP13/14. We have constructed the first-described doubly fluorescence-tagged virus expressing VP22 and VP13/14 as fusion proteins with cyan fluorescent protein and yellow fluorescent protein, respectively. Visualization of both proteins within the same live infected cells has indicated that these two tegument proteins localize to the same nuclear dots but that VP22 appears there earlier than VP13/14. Further studies have shown that these tegument-specific dots are detectable as phase-dense bodies as early as 2 h after infection and that they are different from the previously described nuclear domains that contain capsid proteins. They are also different from the ICP0 domains formed at cellular nuclear domain 10 sites early in infection but, in almost all cases, are located in juxtaposition to these ICP0 domains. Hence, these tegument proteins join a growing number of proteins that are targeted to discrete nuclear domains in the herpesvirus-infected cell nucleus. PMID:12239313

Hutchinson, Ian; Whiteley, Alison; Browne, Helena; Elliott, Gillian




PubMed Central

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





PubMed Central

By means of qualitative staining experiments the characteristic protein changes which occur during maturation of salmon sperm can be followed. It can be observed that the replacement of histone by protamine takes place after completion of meiosis during an advanced stage of spermiogenesis.

Alfert, Max



Analysis of rat liver chromatin and nuclear proteins after nutritional variation 1,2.  


Chemical composition of liver chromatin was determined for rats fed a complete stock diet, or a diet lacking protein or fat. High carbohydrate, fat-free (diet 1) and low carbohydrate, protein-free (diet 2) diets were selected because they elicit structural alteration in chromatin as measured by incubation with micrococcal nuclease (E.C. In the present study, either dietary treatment caused an increase in mass ratios of RNA:DNA and nonhistone:DNA, relative to control ratios. The nonhistone-DNA ratios in liver of rats fed diet 1 or diet 2 were 2.4-fold and 3.5-fold, respectively, larger than control ratios. The histone:DNA ratio remained relatively constant among all three dietary treatments. Liver nuclei were purified from rats fed each dietary treatment and were solubilized in 9 M urea. The nuclear proteins were analyzed by two-dimensional electrophoresis and visualized with a silver treatment that stains proteins in color. The electrophoretograms presented show preferentially proteins with low molecular weights and acidic pIs, two characteristics of nonhistones. The two-dimensional protein patterns are nearly identical for nuclear proteins from all three treatments. Analysis of the electrophoretograms indicates that the diet-induced increased nonhistone:DNA ratios are apparently not attributable to new species of protein, but rather to increased relative abundance of many proteins in the existing populations. PMID:7086547

Castro, C E; Sevall, J S



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.



The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import  

PubMed Central

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin ?/?– or transportin-dependent import.

Walther, Tobias C.; Pickersgill, Helen S.; Cordes, Volker C.; Goldberg, Martin W.; Allen, Terry D.; Mattaj, Iain W.; Fornerod, Maarten



Dynamic Nuclear Polarization Methods in Solids and Solutions to Explore Membrane Proteins and Membrane Systems  

NASA Astrophysics Data System (ADS)

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Cheng, Chi-Yuan; Han, Songi



Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells  

PubMed Central

The C proteins (C1 and C2) are major constituents of the 40S subparticle of heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) (Beyer, A.L., M.E. Christensen, B.W. Walker, and W.M. LeStourgeon, 1977, Cell, 11:127-138) and are two of the most prominent proteins that become cross-linked by ultraviolet light to heterogeneous nuclear RNA (hnRNA) in vivo. Studies are described here on the characterization of the C proteins in vertebrate cells using monoclonal and polyclonal antibodies. Monoclonal antibodies to genuine RNP proteins, including the C proteins, were obtained by immunizing mice with purified complexes of poly(A)+ hnRNA and poly(A)+ mRNA with their contacting proteins in vivo obtained by ultraviolet cross-linking the complexes in intact cells (Dreyfuss, G., Y.D. Choi, and S.A. Adam, 1984, Mol. Cell. Biol., 4:1104-1114). One of the monoclonal antibodies identified the C proteins in widely divergent species ranging from human to lizard. In all species examined, there were two C proteins in the molecular weight range of from 39,000 to 42,000 for C1, and from 40,000 to 45,000 for C2. The two C proteins were found to be highly related to each other; they were recognized by the same monoclonal antibodies and antibodies raised against purified C1 reacted also with C2. In avian, rodent, and human cells the C proteins were phosphorylated and were in contact with hnRNA in vivo. Immunofluorescence microscopy demonstrated that the C proteins are segregated to the nucleus. Within the nucleus the C proteins were not found in nucleoli and were not associated with chromatin as seen in cells in prophase. These findings demonstrate that C proteins with similar characteristics to those in humans are ubiquitous components of hnRNPs in vertebrates.



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



Structurally Related but Functionally Distinct Yeast Sm D Core Small Nuclear Ribonucleoprotein Particle Proteins  

Microsoft Academic Search

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4\\/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We




Expanded Polyglutamine Protein Forms Nuclear Inclusions and Causes Neural Degeneration in Drosophila  

Microsoft Academic Search

Spinocerebellar ataxia type 3 (SCA3\\/MJD) is one of at least eight human neurodegenerative diseases caused by glutamine-repeat expansion. We have recreated glutamine-repeat disease in Drosophila using a segment of the SCA3\\/MJD protein. Targeted expression of the protein with an expanded polyglutamine repeat led to nuclear inclusion (NI) formation and late-onset cell degeneration. Differential sensitivity to the mutant transgene was observed

John M. Warrick; Henry L. Paulson; Gladys L. Gray-Board; Quang T. Bui; Kenneth H. Fischbeck; Randall N. Pittman; Nancy M. Bonini



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



Dimerization and nuclear entry of mPER proteins in mammalian cells  

PubMed Central

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

Yagita, Kazuhiro; Yamaguchi, Shun; Tamanini, Filippo; van der Horst, Gijsbertus T.J.; Hoeijmakers, Jan H.J.; Yasui, Akira; Loros, Jennifer J.; Dunlap, Jay C.; Okamura, Hitoshi



An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium  

SciTech Connect

Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.

Myre, Michael A. [Department of Biology, University of Toronto at Mississauga, Mississauga, Ont. (Canada); O'Day, Danton H. [Department of Biology, University of Toronto at Mississauga, Mississauga, Ont. (Canada)]. E-mail:



Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins.  


GroEL-protein interactions were characterized by stable isotope-assisted nuclear magnetic resonance (NMR) spectroscopy using chemically denatured bovine rhodanese and an intrinsically disordered protein, ?-synuclein, as model ligands. NMR data indicated that proteins tethered to GroEL remain largely unfolded and highly mobile, enabling identification of the interaction hot spots displayed on intrinsically disordered proteins. PMID:23567152

Nishida, Noritaka; Yagi-Utsumi, Maho; Motojima, Fumihiro; Yoshida, Masasuke; Shimada, Ichio; Kato, Koichi



Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome-nucleus association and transcriptional signaling  

PubMed Central

The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2? also have aberrant distributions, but lamin B and LAP2? have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus–centrosome distances, increased ?-catenin signaling, and relocalization of ?-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome–nuclear envelope association and the regulation of ?-catenin transcriptional co-activator activity that is dependent on ?-catenin nuclear export.

Meyer, Adam J.; Almendrala, Donna K.; Go, Minjoung M.; Krauss, Sharon Wald



A GTPase distinct from Ran is involved in nuclear protein import  

PubMed Central

Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.



Ribosome biogenesis factors bind a nuclear envelope SUN domain protein to cluster yeast telomeres  

PubMed Central

Two interacting ribosome biogenesis factors, Ebp2 and Rrs1, associate with Mps3, an essential inner nuclear membrane protein. Both are found in foci along the nuclear periphery, like Mps3, as well as in the nucleolus. Temperature-sensitive ebp2 and rrs1 mutations that compromise ribosome biogenesis displace the mutant proteins from the nuclear rim and lead to a distorted nuclear shape. Mps3 is known to contribute to the S-phase anchoring of telomeres through its interaction with the silent information regulator Sir4 and yKu. Intriguingly, we find that both Ebp2 and Rrs1 interact with the C-terminal domain of Sir4, and that conditional inactivation of either ebp2 or rrs1 interferes with both the clustering and silencing of yeast telomeres, while telomere tethering to the nuclear periphery remains intact. Importantly, expression of an Ebp2–Mps3 fusion protein in the ebp2 mutant suppresses the defect in telomere clustering, but not its defects in growth or ribosome biogenesis. Our results suggest that the ribosome biogenesis factors Ebp2 and Rrs1 cooperate with Mps3 to mediate telomere clustering, but not telomere tethering, by binding Sir4.

Horigome, Chihiro; Okada, Takafumi; Shimazu, Kyoko; Gasser, Susan M; Mizuta, Keiko



Nup214 is required for CRM1-dependent nuclear protein export in vivo.  


Nucleoporins mediate transport of macromolecules across the nuclear pore complex, yet the function of many individual nucleoporins is largely unresolved. To address this question, we depleted cells of the cytoplasmic nucleoporins Nup214/CAN and Nup358/RanBP2 by RNA interference. Depletion of Nup214 resulted in codepletion of its binding partner, Nup88. Nuclear pore complexes assembled in the absence of Nup214/Nup88 or Nup358 were fully functional in nuclear protein import, whereas nuclear mRNA export was slightly impaired. Depletion of Nup358 had only a minor effect on nuclear protein export. In contrast, depletion of Nup214/Nup88 led to strongly reduced CRM1-mediated export of the shuttling transcription factor NFAT as well as a human immunodeficiency virus-Rev derivative. A specific role of Nup214 in protein export is furthered by the biochemical properties of a high-affinity complex containing Nup214, CRM1, RanGTP, and an export cargo. Our results show that the Nup214/Nup88 complex is required for efficient CRM1-mediated transport, supporting a model involving a high-affinity binding site for CRM1 at Nup214 in the terminal steps of export. PMID:16943420

Hutten, Saskia; Kehlenbach, Ralph H



The Leukocyte Nuclear Envelope Proteome Varies with Cell Activation and Contains Novel Transmembrane Proteins That Affect Genome Architecture*  

PubMed Central

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

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



Nuclear Localization Signal(s) Required for Nuclear Targeting of the Maize Regulatory Protein Opaque2  

Microsoft Academic Search

The maize regulatory protein Opaque-2 (02) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of 02 to reporter protein p-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of 02 containing 135 and 149 amino

Marguerite J. Varagona; Robert J. Schmidt; Natasha V. Raikhelai



Rat Liver Nuclear Nonhistone Proteins: A Partial Fractionation and Their Interaction with DNA.  

National Technical Information Service (NTIS)

Rat liver nuclear proteins can be separated into two nonhistone fractions and one histone fraction on Bio Rex 70 exp + columns in the presence of urea. Rechromatography of the combined nonhistone peaks on Bio Rex 70 exp + yields four fractions. One of the...

D. M. Sheehan



Nonhistone nuclear high mobility group proteins 14 and 17 stabilize nucleosome core particles  

Microsoft Academic Search

Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of

A. E. Paton; E. Wilkinson-Singley; D. W. Olins



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



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:



Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins  

PubMed Central

Although the nuclear envelope is a dynamic structure that disassembles and reforms during mitosis, the formation of membranous vesicles derived from the nuclear envelope has not yet been described in noninfected cells. However, during herpesvirus maturation, intranuclear capsids initiate transit to the cytosol for final maturation by budding at the inner nuclear membrane. Two conserved herpesvirus proteins are required for this primary envelopment, designated in the alphaherpesviruses as pUL31 and pUL34. Here, we show that simultaneous expression of pUL31 and pUL34 of the alphaherpesvirus pseudorabies virus in stably transfected rabbit kidney cells resulted in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid. They contain pUL31 and pUL34 as shown by immunolabeling and are derived from the nuclear envelope. Thus, coexpression of only two conserved herpesvirus proteins without any other viral factor is sufficient to induce the formation of vesicles from the nuclear membrane. This argues for the contribution of cellular factors in this process either recruited from their natural cytoplasmic location or not yet identified as components of the nuclear compartment.

Klupp, Barbara G.; Granzow, Harald; Fuchs, Walter; Keil, Gunther M.; Finke, Stefan; Mettenleiter, Thomas C.



Protocols for nuclei isolation and nuclear protein extraction from the resurrection plant Xerophyta viscosa for proteomic studies  

Microsoft Academic Search

The plant nucleus is an important subcellular organelle but the isolation of pure and enriched nuclei from plants and subsequent extraction of nuclear proteins for proteomic studies is challenging. Here, we present protocols for nuclei isolation and nuclear protein extraction from the resurrection plant, Xerophyta viscosa, and show optimization and modification of the most critical steps.

Kamal Omer Abdalla; Jennifer Ann Thomson; Muhammad Suhail Rafudeen



[Nuclear organization and expression of milk protein genes].  


Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products. PMID:17151554

Chanat, Eric; Aujean, Etienne; Balteanu, Adrian; Chat, Sophie; Coant, Nicolas; Fontaine, Marie-Louise; Hue-Beauvais, Cathy; Péchoux, Christine; Torbati, Mohammad Bagher Montazer; Pauloin, Alain; Petitbarat, Marie; Devinoy, Eve



Nuclear proteins in Drosophila melanogaster cells after heat shock and their binding to homologous DNA.  

PubMed Central

After 5 minutes heat shock at 37 degrees C Drosophila melanogaster Kc-cell nuclear proteins were extracted wit 0.4M NaCl and compared by SDS gel electrophoresis with extracts from cells grown at 25 degrees C. Two proteins (39 000 and 46 000) were only found in heat shock nuclei. Reconstitution with total Drosophila DNA or a DNA fragment from the heat inducible locus 87A/C covalently coupled to sepharose was performed. In the presence of calf thymus competitor DNA these proteins and also others of lower molecular weight showed preferential binding to the homologous DNA. Images

Falkner, F G; Biessmann, H



Characterization of the nuclear localization signal of the mouse TET3 protein.  


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-?. PMID:23998935

Xiao, Peng; Zhou, Xiao-Long; Zhang, Hong-Xiao; Xiong, Kai; Teng, Yun; Huang, Xian-Ju; Cao, Rui; Wang, Yi; Liu, Hong-Lin



Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.  


Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (?-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaC?), and Scnn1g (ENaC?), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in ?-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics ( and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct. PMID:22440904

Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A



Strategies to inhibit viral protein nuclear import: HIV-1 as a target  

PubMed Central

Nuclear import is a critical step in the life cycle of HIV-1. During the early (pre-integration) stages of infection, HIV-1 has to transport its pre-integration complex into the nucleus for integration into the host cell chromatin, while at the later (post-integration) stages viral regulatory proteins Tat and Rev need to get into the nucleus to stimulate transcription and regulate splicing and nuclear export of subgenomic and genomic RNAs. Given such important role of nuclear import in HIV-1 life cycle, this step presents an attractive target for anti-viral therapeutic intervention. In this review, we describe the current state of our understanding of the interactions regulating nuclear import of the HIV-1 pre-integration complex and describe current approaches to inhibit it.

Levin, Aviad; Loyter, Abraham; Bukrinsky, Michael



The nuclear localization signal of the NS1 protein is essential for Periplaneta fuliginosa densovirus infection.  


The regulatory protein NS1 is a key molecule in life cycle of Periplaneta fuliginosa densovirus (PfDNV). When we ectopically expressed the PfDNV NS1 protein in non-P. fuliginosa insect cells, the NS1 protein could not enter the nucleus and remained in the cytosol. However, the NS1 was localized to both the cytosol and nucleus of cockroach hemocyte cells. So we investigated the abilities of the potential nuclear localization signal (NLS) of P. fuliginosa Densovirus non-structural protein 1 (NS1) to translocate NS1 and a carrier protein to the nucleus following transfection into insect cells. Possible nuclear localization sequences were chosen from the NS1 on the basis of the presence of basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear localization activity was found within the residues 252-257 (RRRRRR) of the NS1, while replacement of a single arginine in this region with glycine abolished it. The targeting activity was enhanced with the arginine residues added. PMID:19596391

Zhou, Weidong; Zhang, Jiamin; Yang, Bo; Zhou, Liang; Hu, Yuanyang



Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase  

PubMed Central

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

Moilanen, Anu-Maarit; Karvonen, Ulla; Poukka, Hetti; Janne, Olli A.; Palvimo, Jorma J.



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

Microsoft Academic Search

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 mi- grate on SDS-PAGE as 17- and 52-kDa

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


Roles of LAP2 Proteins in Nuclear Assembly and DNA Replication: Truncated LAP2beta Proteins Alter Lamina Assembly, Envelope Formation, Nuclear Size, and DNA Replication Efficiency in Xenopus laevis Extracts  

Microsoft Academic Search

Humans express three major splicing iso- forms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2 b and g are integral membrane proteins, whereas a is intranuclear. When truncated recombinant human LAP2 b proteins were added to cell-free Xeno- pus laevis nuclear assembly reactions at high concentra- tions, a domain common to all LAP2 isoforms (residues 1-187) inhibited membrane binding

Tracey Michele Gant; Crafford A. Harris; Katherine L. Wilson



Nuclear localization of orphan receptor protein kinase (Ror1) is mediated through the juxtamembrane domain  

PubMed Central

Background Several receptor tyrosine kinases (RTKs) such as EGFR, FGFR, TRK, and VEGFR are capable of localizing in the cell nucleus in addition to their usual plasma membrane localization. Recent reports also demonstrate that nuclear-localized RTKs have important cellular functions such as transcriptional activation. On the basis of preliminary bioinformatic analysis, additional RTKs, including receptor tyrosine kinase-like orphan receptor 1 (Ror1) were predicted to have the potential for nuclear subcellular localization. Ror1 is a receptor protein tyrosine kinase that modulates neurite growth in the central nervous system. Because the nuclear localization capability of the Ror1 cytoplasmic domain has not been reported, we examined the cellular expression distribution of this region. Results The Ror1 cytoplasmic region was amplified and cloned into reporter constructs with fluorescent tags. Following transfection, the nuclear distribution patterns of transiently expressed fusion proteins were observed. Serial deletion constructs were then used to map the juxtamembrane domain of Ror1 (aa_471-513) for this nuclear translocation activity. Further site-directed mutagenesis suggested that a KxxK-16 aa-KxxK sequence at residues 486-509 is responsible for the nuclear translocation interaction. Subsequent immunofluorescence analysis by cotransfection of Ran and Ror1 implied that the nuclear translocation event of Ror1 might be mediated through the Ran pathway. Conclusions We have predicted several RTKs that contain the nuclear localization signals. This is the first report to suggest that the juxtamembrane domain of the Ror1 cytoplasmic region mediates the translocation event. Ran GTPase is also implicated in this event. Our study might be beneficial in future research to understand the Ror1 biological signaling pathway.



The Simian virus 40 late viral protein VP4 disrupts the nuclear envelope for viral release.  


Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release. PMID:22238309

Giorda, Kristina M; Raghava, Smita; Hebert, Daniel N



Nuclear Import of the Stem-Loop Binding Protein and Localization during the Cell Cycle  

PubMed Central

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem–loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Imp?/Imp? and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Imp?/Imp? binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Imp?/Imp? pathway contributes to SLBP nuclear import in HeLa cells.

Erkmann, Judith A.; Wagner, Eric J.; Dong, Jian; Zhang, Yanping; Kutay, Ulrike; Marzluff, William F.



Protein folding at atomic resolution: analysis of autonomously folding supersecondary structure motifs by nuclear magnetic resonance.  


The study of protein folding has been conventionally hampered by the assumption that all single-domain proteins fold by an all-or-none process (two-state folding) that makes it impossible to resolve folding mechanisms experimentally. Here we describe an experimental method for the thermodynamic analysis of protein folding at atomic resolution using nuclear magnetic resonance (NMR). The method is specifically developed for the study of small proteins that fold autonomously into basic supersecondary structure motifs, and that do so in the sub-millisecond timescale (folding archetypes). From the NMR experiments we obtain hundreds of atomic unfolding curves that are subsequently analyzed leading to the determination of the characteristic network of folding interactions. The application of this approach to a comprehensive catalog of elementary folding archetypes holds the promise of becoming the first experimental approach capable of unraveling the basic rules connecting protein structure and folding mechanism. PMID:22987355

Sborgi, Lorenzo; Verma, Abhinav; Sadqi, Mourad; de Alba, Eva; Muñoz, Victor



The nuclear pore complex protein Nup88 is overexpressed in tumor cells.  


It has been previously shown (J. Schneider et al., Br. J. Cancer, 77: 1015-1020, 1998) that an antibody directed against Candida albicans proteins (C6) recognizes specifically a protein in human ovarian tumors. We have now performed an immunoscreening of a human cDNA library using this antibody and identified the antigen as Nup88, a protein localized preferentially at the nuclear membrane and probably implicated in nucleocytoplasmic transport. Our results show that Nup88 is strongly expressed in a series of human tumor cell lines compared with nontransformed cell lines at the RNA and the protein levels. Furthermore, we observed that in 76% of 21 ovarian tumors analyzed, the protein is also overexpressed in malignant tissue when compared with healthy adjacent tissue. Nup88 may, therefore, be considered as a putative marker for tumor growth and is probably related to increased cell cycling. PMID:10554006

Martínez, N; Alonso, A; Moragues, M D; Pontón, J; Schneider, J



The characterization of the nuclear dynamics of syntenin-2, a PIP2 binding PDZ protein.  


Cellular signaling is largely dependent on the presence, that is, assembly/disassembly, of supramolecular complexes. Postsynaptic density protein, Discs-large, Zona occludens (PDZ) domains play important roles in the assembly of various signaling complexes. Syntenin-2 (S2) is a PDZ protein that interacts with nuclear phosphatidylinositol 4,5-bisphosphate (PIP2 ). Although nuclear lipids emerge as key players in nuclear processes, the global significance of nuclear phosphoinositide-protein interactions is still poorly understood. Those phosphoinositide-protein interactions that have been studied in detail appear to have profound physiological effects. To our knowledge none of these were investigated by dynamic studies such as Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), or Fluorescence Recovery After Photobleaching (FRAP). Although the exact function of S2 is unknown, siRNA experiments suggest that this PDZ protein plays a role in the organization of nuclear PIP2 , cell division, and cell survival. As a consequence of its PIP2 interaction, its reported self-association in a yeast two-hybrid study and its speculated interaction with many, yet unidentified, proteins one can hypothesize that S2 plays an important role in cell signaling. Therefore, we studied the dynamics of S2 using FCS, FCCS, and FRAP, utilizing an active truncated form deleted for the first 94 amino acids (S2-?N). We showed that S2-?N self-associates and is distributed in three groups. One immobile group, one slow diffusing group, which interacts with the nuclear environment and one fast diffusing group, which is not incorporated in high molecular weight complexes. In addition, our FCS and FRAP measurements on S2-?N mutants affected in their PIP2 binding showed that PIP2 plays an important role in the distribution of S2-?N among these groups, and favors the enrichment of S2-?N in the slow diffusing and immobile group. This work indicates that S2 relies on nuclear PIP2 to interact with practically immobile structures, possibly chromatin. © 2013 International Society for Advancement of Cytometry. PMID:23300061

Geeraerts, Annelies; Hsiu-Fang, Fan; Zimmermann, Pascale; Engelborghs, Yves



Proteomics profiling of nuclear proteins for kidney fibroblasts suggests hypoxia, meiosis, and cancer may meet in the nucleus.  


Proteomics methods were used to characterize proteins that change their form or abundance in the nucleus of NRK49F rat kidney fibroblasts during prolonged hypoxia (1% O(2), 12 h). Of the 791 proteins that were monitored, about 20% showed detectable changes. The 51 most abundant proteins were identified by mass spectrometry. Changes in nuclear receptor transcription factors (THRalpha1, RORalpha4, HNF4alpha, NUR77), other transcription factors (GATA1, AP-2alpha, OCT1, ATF6alpha, ZFP161, ZNF354A, PDCD2), and transcription cofactors (PC4, PCAF, MTA1, TCEA1, JMY) are indicative of major, co-ordinated changes in transcription. Proteins involved in DNA repair/recombination, ribosomal RNA synthesis, RNA processing, nuclear transport, nuclear organization, protein translation, glycolysis, lipid metabolism, several protein kinases (PKCdelta, MAP3K4, GRK3), as well as proteins with no established functional role were also observed. The observed proteins suggest nuclear regulatory roles for proteins involved in cytosolic processes such as glycolysis and fatty acid metabolism, and roles in overall nuclear structure/organization for proteins previously associated with meiosis and/or spermatogenesis (synaptonemal complex proteins 1 and 2 (SYCP1, SYCP2), meiosis-specific nuclear structural protein 1 (MNS1), LMNC2, zinc finger protein 99 (ZFP99)). Proteins associated with cytoplasmic membrane functions (ACTN4, hyaluronan mediated motility receptor (RHAMM), VLDLR, GRK3) and/or endocytosis (DNM2) were also seen. For 30% of the identified proteins, new isoforms indicative of alternative transcription were detected (e.g., GATA1, ATF6alpha, MTA1, MLH1, MYO1C, UBF, SYCP2, EIF3S10, MAP3K4, ZFP99). Comparison with proteins involved in cell death, cancer, and testis/meiosis/spermatogenesis suggests commonalities, which may reflect fundamental mechanisms for down-regulation of cellular function. PMID:15942958

Shakib, Kaveh; Norman, Jill T; Fine, Leon G; Brown, Larry R; Godovac-Zimmermann, Jasminka



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:; 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)



Protein-Protein Interactions during Bacterial Chemotaxis using Methyl TROSY Nuclear Magnetic Resonance.  

NASA Astrophysics Data System (ADS)

During bacterial chemotaxis, the histidine autokinase CheA interacts with the chemotaxis receptors with the help of the coupling protein CheW. The CheA-CheW interaction is typical of many macromolecular complexes where protein-protein interactions play an important role. In this case a relatively small protein, CheW (18 kDalton), becomes part of a much larger complex. Here we describe a new method to map the residues at a protein-protein interface for macromolecular complexes of molecular weight greater than 100 kDalton. The method exploits the C13 methyl TROSY methodology developed in Lewis Kay's laboratory. The essence of the Kay approach is that a portion of the intensity of HMQC spectra of individual -(13)CH3 resonances in an otherwise deuterated macromolecule have much reduced dipole-dipole relaxation and remain sharp and relatively easy to detect , even in macromolecules of molecular mass 100 kD or greater. The reduction in dipolar interactions is lost if a given methyl group comes in close contact with other protons such as those supplied by the interface of a protonated interaction partner. Comparing the -(13)CH3 resonances of a protein of interest in the presence of a protonated versus deuterated interaction partner allows the methyls at the interface can be identified. The application of the approach for establishing points of contact between CheA and CheW will be discussed.

Hamel, Damon; Dahlquist, Frederick



The nuclear matrix protein NMP-1 is the transcription factor YY1.  

PubMed Central

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6

Guo, B; Odgren, P R; van Wijnen, A J; Last, T J; Nickerson, J; Penman, S; Lian, J B; Stein, J L; Stein, G S



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)



Nuclear contour irregularity and abnormal transporter protein distribution in anterior horn cells in amyotrophic lateral sclerosis.  


The nucleocytoplasmic transport system is essential for maintaining cell viability; transport of proteins and nucleic acids between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). In this study, we examined the immunohistochemical distribution of the major protein components of NPCs, Nup62, Nup88, and Nup153, in spinal cords from controls and patients with sporadic or familial amyotrophic lateral sclerosis (SALS or FALS) and its mouse model. In control subjects, immunolabeling on the nuclear envelopes of anterior horn cells (AHCs) was invariably smooth and continuous, whereas in SALS and FALS patients, the AHCs predominantly showed irregular nuclear contours. Double immunofluorescence staining demonstrated that in SALS patients, importin-beta immunoreactivity was absent in the nuclei in a subset of AHCs; in these cells, Nup62 immunolabeling of nuclear membrane was invariably irregular, suggesting that there was dysfunctional nucleocytoplasmic transport in those AHCs. In the mouse model, Nup62-immunolabeled AHCs with irregular nuclear contours were predominant as early as the presymptomatic stage and the contours became progressively discontinuous along with disease development. Together, these observations suggest that dysfunctional nucleocytoplasmic transport may underlie the pathogenesis of ALS. PMID:19816199

Kinoshita, Yoshimi; Ito, Hidefumi; Hirano, Asao; Fujita, Kengo; Wate, Reika; Nakamura, Masataka; Kaneko, Satoshi; Nakano, Satoshi; Kusaka, Hirofumi



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


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



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


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



Arabidopsis WPP-domain proteins are developmentally associated with the nuclear envelope and promote cell division.  


The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference-based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity. PMID:15548735

Patel, Shalaka; Rose, Annkatrin; Meulia, Tea; Dixit, Ram; Cyr, Richard J; Meier, Iris



The MCM-associated protein MCM-BP is important for human nuclear morphology.  


Mini-chromosome maintenance complex-binding protein (MCM-BP) was discovered as a protein that is strongly associated with human MCM proteins, known to be crucial for DNA replication in providing DNA helicase activity. The Xenopus MCM-BP homologue appears to play a role in unloading MCM complexes from chromatin after DNA synthesis; however, the importance of MCM-BP and its functional contribution to human cells has been unclear. Here we show that depletion of MCM-BP by sustained expression of short hairpin RNA (shRNA) results in highly abnormal nuclear morphology and centrosome amplification. The abnormal nuclear morphology was not seen with depletion of other MCM proteins and was rescued with shRNA-resistant MCM-BP. MCM-BP depletion was also found to result in transient activation of the G2 checkpoint, slowed progression through G2 and increased replication protein A foci, indicative of replication stress. In addition, MCM-BP depletion led to increased cellular levels of MCM proteins throughout the cell cycle including soluble MCM pools. The results suggest that MCM-BP makes multiple contributions to human cells that are not limited to unloading of the MCM complex. PMID:22250201

Jagannathan, Madhav; Sakwe, Amos M; Nguyen, Tin; Frappier, Lori



Internalization and kinetics of nuclear migration of protein-only, arginine-rich nanoparticles.  


Understanding the intracellular trafficking of nanoparticles internalized by mammalian cells is a critical issue in nanomedicine, intimately linked to therapeutic applications but also to toxicity concerns. While the uptake mechanisms of carbon nanotubes and polymeric particles have been investigated fairly extensively, there are few studies on the migration and fate of protein-only nanoparticles other than natural viruses. Interestingly, protein nanoparticles are emerging as tools in personalized medicines because of their biocompatibility and functional tuneability, and are particularly promising for gene therapy and also conventional drug delivery. Here, we have investigated the uptake and kinetics of intracellular migration of protein nanoparticles built up by a chimerical multifunctional protein, and functionalized by a pleiotropic, membrane-active (R9) terminal peptide. Interestingly, protein nanoparticles are first localized in endosomes, but an early endosomal escape allows them to reach and accumulate in the nucleus (but not in the cytoplasm), with a migration speed of 0.0044 ± 0.0003 ?m/s, ten-fold higher than that expected for passive diffusion. Interestingly, the plasmatic, instead of the nuclear membrane is the main cellular barrier in the nuclear way of R9-assisted protein-only nanoparticles. PMID:20869766

Vázquez, Esther; Cubarsi, Rafael; Unzueta, Ugutz; Roldán, Mónica; Domingo-Espín, Joan; Ferrer-Miralles, Neus; Villaverde, Antonio



Pml39, a Novel Protein of the Nuclear Periphery Required for Nuclear Retention of Improper Messenger RibonucleoparticlesD?  

PubMed Central

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60? mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39? partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.

Palancade, Benoit; Zuccolo, Michela; Loeillet, Sophie; Nicolas, Alain; Doye, Valerie



Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a dynamic nuclear and sarcomeric protein  

PubMed Central

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1's role in vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular pools of FRG1 are linked. During myoblast differentiation, FRG1's subcellular distribution changed dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD.

Hanel, Meredith L.; Sun, Chia-Yun Jessica; Jones, Takako I.; Long, Steven W.; Zanotti, Simona; Milner, Derek; Jones, Peter L.



Nuclear effects: unexpected intracellular actions of insulin-like growth factor binding protein-3.  


Insulin-like growth factor (IGF) binding protein (IGFBP)-3 has been shown to be a growth inhibitory, apoptosis-inducing molecule by virtue of its ability to bind IGFs, in addition to previously demonstrated IGF-independent effects. The recent discovery of the interaction between nuclear IGFBP-3 and 9-cis retinoic acid receptor-alpha (retinoid X receptor alpha RXRalpha), a nuclear receptor, and its involvement in the regulation of transcriptional signaling and apoptosis represents an important paradigm shift in the understanding of IGFBP function. RXRalpha is required for the apoptosis-inducing effects of IGFBP-3. IGFBP-3 and RXR ligands are additive in inducing apoptosis in cancer cells. IGFBP-3 has direct effects on gene transcription, as RXR response element reporter signaling was enhanced and the all-trans retinoic acid receptor response element reporter signaling was inhibited. Accumulating evidence further confirms IGF-independent functions of this multifunction binding protein. Other binding proteins, in addition to other members of the IGF axis, have now been described in the nucleus and are postulated to have effects on transcriptional events. Investigation into these new interactions will expose new protein partners in the interface between the nuclear receptor and growth factor pathways and reveal new targets to be exploited in the treatment of cancer and other diseases. PMID:12379488

Lee, K-W; Cohen, P



The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site.  


RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52. PMID:20190268

Saito, Kengo; Kagawa, Wataru; Suzuki, Takehiro; Suzuki, Hidekazu; Yokoyama, Shigeyuki; Saitoh, Hisato; Tashiro, Satoshi; Dohmae, Naoshi; Kurumizaka, Hitoshi



Nuclear translocation of small G protein RhoA via active transportation in gastric cancer cells.  


Recent studies have shown the localization of RhoA in the cell nucleus, in addition to its cellular distribution in the cytosol and cell membrane. Our previous results that a high amount of RhoA was detected in gastric cancer cell nucleus and application of anticancer drug Taxol could reduce RhoA nuclear localization, suggest a relationship between nuclear translocation of RhoA and tumor progression. However, the mechanism and biological function of RhoA nuclear translocation remain unclear. The aim of the present study was to explore the mole-cular mechanism(s) for RhoA protein entering the nucleus in the human gastric cancer cell line AGS. Using immunofluorescence microscopy we observed not only a partial colocalization of RhoA with importin ?, mainly surrounding and on the nuclear membrane, but also an intensive colocalization of RhoA with NF-?B P50 in both cytoplasm and nucleus, particularly in the cell nucleoli. A strong association between RhoA and importin ? as well as RhoA and NF-?B P50 was revealed by co-immunoprecipitation and western blotting. Moreover, AGS cells treated with the nuclear export inhibitor, leptomycin B (LMB), showed an increase of RhoA protein amount in the nucleus, indicating an active transport process for nuclear export of RhoA. Taken together, our results suggest that nuclear translocation of RhoA in AGS cells is via active transportation, a process through importin ? in a NF-?B-dependent manner. PMID:23900609

Xu, Jin; Li, Yueying; Yang, Xiaoming; Chen, Yongchang; Chen, Min



Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus  

Microsoft Academic Search

RNA synthesis (genome replication and subgenomic mRNA transcription) directed by equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) occurs on modified cytoplasmic membranes to which most viral replicase subunits localize. Re- markably, a fraction of non-structural protein 1 (nsp1), a protein essential for transcription but dis- pensable for genome replication, is present in the host cell nucleus, in particular during

Marieke A. Tijms; Yvonne van der Meer; Eric J. Snijder


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

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



Importin-? Protein Binding to a Nuclear Localization Signal of Carbohydrate Response Element-Binding Protein (ChREBP)*  

PubMed Central

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. Circulating blood glucose levels affect ChREBP activity in hepatocytes largely by post-translational mechanisms that include phosphorylation-dependent subcellular localization. Previously, we showed that ChREBP is retained in the cytosol by phosphorylation-dependent binding to 14-3-3 protein dimers and identified the ?2 helix (residues 125–135) phospho-Ser140 domain as the primary 14-3-3 binding site (Sakiyama, H., Wynn, R. M., Lee, W. R., Fukasawa, M., Mizuguchi, H., Gardner, K. H., Repa, J. J., and Uyeda, K. (2008) J. Biol. Chem. 283, 24899–24908). To enter the nucleus in response to high glucose, ChREBP must bind importin-?; this heterodimer then forms a complex with importin-? to interact with the nuclear pore complex. In this work, we recharacterized the importin-? binding nuclear localization signal (NLS) of rat ChREBP, identifying it as an extended classical bipartite NLS encompassing minimally residues 158–190. Replacing Lys159/Lys190 residues of ChREBP with alanine resulted in loss of importin-? binding, glucose-stimulated transcriptional activity and nuclear localization. A secondary 14-3-3 protein binding site also was identified, the ?3 helix (residues 170–190) phospho-Ser196 domain. Importin-? and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-? and 14-3-3 control movement of ChREBP in and out of the nucleus in response to changes in glucose levels in liver and thus further suggest that the extended NLS of ChREBP is a critical glucose-sensing, glucose-responsive site.

Ge, Qiang; Nakagawa, Tsutomu; Wynn, R. Max; Chook, Yuh Min; Miller, Bonnie C.; Uyeda, Kosaku



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

PubMed Central

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

Bresson, Stefan M.; Conrad, Nicholas K.



Nuclear expression of Smad proteins and its prognostic significance in clear cell renal cell carcinoma.  


Smad2, Smad3, and Smad4 are components of the transforming growth factor ? signaling pathway associated with tumorigenesis. The expression of these proteins is associated with tumor progression and prognosis of many cancers. This study aimed to evaluate the nuclear expression of Smad2, Smad3, and Smad4 in clear cell renal cell carcinoma and to assess the clinical significance and prognostic value of their expression patterns. The nuclear expression levels of Smads were evaluated in 637 cases of clear cell renal cell carcinomas using immunohistochemistry. To determine the statistical significance of Smad expression in clear cell renal cell carcinoma, each of the cases were divided into 2 groups (low and high expression groups) according to the extent of nuclear staining. Nuclear expressions of Smad3 and Smad4 were inversely correlated with the patient's age, the nuclear grade, the tumor size, and the pTNM stage. The Smad3-low and Smad4-low groups showed significantly shorter cancer-specific and progression-free survival times. Furthermore, multivariate analysis showed that both Smad3 and Smad4 were independent predictors for progression-free survival (P = .008 and P = .022, respectively). However, Smad2 expression was not related to clinicopathologic parameters and patients' survival. These results suggest that nuclear expressions of Smad3 and Smad4 were related to prognosis of clear cell renal cell carcinoma patients and may serve as novel prognostic markers in clear cell renal cell carcinoma patients. PMID:23668999

Park, Jeong Hwan; Lee, Cheol; Suh, Ja Hee; Chae, Ji Youn; Moon, Kyung Chul



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


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

Bresson, Stefan M; Conrad, Nicholas K



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


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

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



A Cytosolic Activity Distinct from Crm1 Mediates Nuclear Export of Protein Kinase Inhibitor in Permeabilized Cells  

Microsoft Academic Search

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes

James M. Holaska; Bryce M. Paschal



Identification of a functional nuclear export signal in the green fluorescent protein asFP499  

SciTech Connect

The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified {sub 194}LRMEKLNI{sub 201} as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES.

Mustafa, Huseyin [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia)]. E-mail:; Strasser, Bernd [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia); Rauth, Sabine [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia); Irving, Robert A. [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia); Wark, Kim L. [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia)



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.

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



Study of reorganization of protein composition of hepatoma cell nuclear matrix during inhibition of DNA synthesis by novobiocin  

SciTech Connect

The nuclear matrix of Zajdela hepatoma cells, in which DNA synthesis was inhibited by novobiocin, contained 2.5-3.0 times more DNA and protein not dissociating in 2 M NaCl than the nuclear matrix from the control cells. Tightly bound DNA-protein complexes not dissociating in 8 M urea or 0.1% SDS were isolated by chromatography of preparations of the nuclear matrix on Sepharose 2B-CL. Subsequent fractionation of the DNA-protein complexes on hydroxyapatite using an eluting buffer containing 4 M guanidine hydrochloride and 5 M urea resulted in partial dissociation of these complexes. An electrophoretic analysis revealed significant changes in the protein composition of the DNA-protein complexes of the hepatoma cell nuclear matrix when DNA synthesis is inhibited.

Gaziev, A.I.; Kutsyi, M.P.



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:; 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)



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:



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:



Effects of the acute myeloid leukemia--associated fusion proteins on nuclear architecture.  


Acute myeloid leukemias (AMLs) are consistently associated with chromosomal rearrangements that result in the generation of chimeric genes and fusion proteins. One of the two affected genes is frequently a transcription factor Involved in the regulation of hematopoletic differentiation. Recent findings suggest a common leukemogenic mechanism for the fused transcription factor: abnormal recruitment of histone deacetylase (HDAC)-containing complexes to its target promoters. Inhibition of HDAC enzymatic activity reverts the leukemic phenotype in vitro and therefore represents a plausible strategy for antileukemic therapy. In this review, we first briefly describe the molecular structure and mechanisms of the most frequent AML associated fusion proteins (RAR, MLL, and CBF fusions) and then summarize available knowledge about their effects on the nuclear architecture. We propose that alteration of nuclear compartmentalization might represent an additional common mechanism of leukemogenesis. PMID:11172539

Faretta, M; Di Croce, L; Pelicci, P G



Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy.  


The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ?30 different proteins, termed nucleoporins, whose arrangement within the NPC is an important unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133-Nup107 in which their long axes are approximately parallel to the nuclear envelope plane. The method we developed can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and may be able to monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines. PMID:21499242

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



Histone H3 Interacts and Colocalizes with the Nuclear Shuttle Protein and the Movement Protein of a Geminivirus ? †  

PubMed Central

Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata.

Zhou, Yanchen; Rojas, Maria R.; Park, Mi-Ri; Seo, Young-Su; Lucas, William J.; Gilbertson, Robert L.



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

Microsoft Academic Search

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

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



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

Microsoft Academic Search

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

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



Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle  

Microsoft Academic Search

In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of non-coding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of small nuclear ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs

Holger Stark; Prakash Dube; Reinhard Lührmann; Berthold Kastner



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



Protein-losing enteropathy associated with hypocomplementemia and anti-nuclear antibodies  

Microsoft Academic Search

:   We report a case of protein-losing enteropathy associated with an autoimmune disorder, presumably systemic lupus erythematosus.\\u000a Although typical manifestations of systemic lupus erythematosus were lacking, a high serum cholesterol level, a low serum\\u000a complement level, positivity for anti-nuclear antibody, and positivity for anti-single-strand DNA antibody suggested an autoimmune\\u000a mechanism as the cause of the condition. Although immunohistological examination of

Atsushi Nakajima; Shin Ohnishi; Toshihide Mimura; Kanae Kubo; Atsushi Suzuki; Yoshio Yazaki; Nobuyuki Matsuhashi



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


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)



Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2  

Microsoft Academic Search

Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that con- tribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of

Stephanie A. Montgomery; Robert E. Johnston



LMNA variants cause cytoplasmic distribution of nuclear pore proteins in Drosophila and human muscle  

PubMed Central

Mutations in the human LMNA gene, encoding A-type lamins, give rise to laminopathies, which include several types of muscular dystrophy. Here, heterozygous sequence variants in LMNA, which result in single amino-acid substitutions, were identified in patients exhibiting muscle weakness. To assess whether the substitutions altered lamin function, we performed in vivo analyses using a Drosophila model. Stocks were generated that expressed mutant forms of the Drosophila A-type lamin modeled after each variant. Larvae were used for motility assays and histochemical staining of the body-wall muscle. In parallel, immunohistochemical analyses were performed on human muscle biopsy samples from the patients. In control flies, muscle-specific expression of the wild-type A-type lamin had no apparent affect. In contrast, expression of the mutant A-type lamins caused dominant larval muscle defects and semi-lethality at the pupal stage. Histochemical staining of larval body wall muscle revealed that the mutant A-type lamin, B-type lamins, the Sad1p, UNC-84 domain protein Klaroid and nuclear pore complex proteins were mislocalized to the cytoplasm. In addition, cytoplasmic actin filaments were disorganized, suggesting links between the nuclear lamina and the cytoskeleton were disrupted. Muscle biopsies from the patients showed dystrophic histopathology and architectural abnormalities similar to the Drosophila larvae, including cytoplasmic distribution of nuclear envelope proteins. These data provide evidence that the Drosophila model can be used to assess the function of novel LMNA mutations and support the idea that loss of cellular compartmentalization of nuclear proteins contributes to muscle disease pathogenesis.

Dialynas, George; Flannery, Kaitlin M.; Zirbel, Luka N.; Nagy, Peter L.; Mathews, Katherine D.; Moore, Steven A.; Wallrath, Lori L.



LMNA variants cause cytoplasmic distribution of nuclear pore proteins in Drosophila and human muscle.  


Mutations in the human LMNA gene, encoding A-type lamins, give rise to laminopathies, which include several types of muscular dystrophy. Here, heterozygous sequence variants in LMNA, which result in single amino-acid substitutions, were identified in patients exhibiting muscle weakness. To assess whether the substitutions altered lamin function, we performed in vivo analyses using a Drosophila model. Stocks were generated that expressed mutant forms of the Drosophila A-type lamin modeled after each variant. Larvae were used for motility assays and histochemical staining of the body-wall muscle. In parallel, immunohistochemical analyses were performed on human muscle biopsy samples from the patients. In control flies, muscle-specific expression of the wild-type A-type lamin had no apparent affect. In contrast, expression of the mutant A-type lamins caused dominant larval muscle defects and semi-lethality at the pupal stage. Histochemical staining of larval body wall muscle revealed that the mutant A-type lamin, B-type lamins, the Sad1p, UNC-84 domain protein Klaroid and nuclear pore complex proteins were mislocalized to the cytoplasm. In addition, cytoplasmic actin filaments were disorganized, suggesting links between the nuclear lamina and the cytoskeleton were disrupted. Muscle biopsies from the patients showed dystrophic histopathology and architectural abnormalities similar to the Drosophila larvae, including cytoplasmic distribution of nuclear envelope proteins. These data provide evidence that the Drosophila model can be used to assess the function of novel LMNA mutations and support the idea that loss of cellular compartmentalization of nuclear proteins contributes to muscle disease pathogenesis. PMID:22186027

Dialynas, George; Flannery, Kaitlin M; Zirbel, Luka N; Nagy, Peter L; Mathews, Katherine D; Moore, Steven A; Wallrath, Lori L



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


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. PMID:8641292

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



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


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



The DNA damage-inducible C. elegans tankyrase is a nuclear protein closely linked to chromosomes.  


Tankyrases are protein members of the poly(ADP-ribose) polymerase family bearing several ankyrin domain and a WGR domain. They have functional role in telomere maintenance, are found at centrosome, and are associated with vesicular secretion. This diversity in localization and function makes it difficult to identify a unified role for tankyrases. We have shown that the C. elegans orthologue PME-5 is among the most transcriptionally up-regulated genes following ionizing radiations, linking a tankyrase with DNA damage response. Our analysis showed that the up-regulation of PME-5 is translated at the protein level, suggesting an effective role in DNA damage response or DNA repair. In order to gain more information on the potential role of PME-5 in DNA damage response, we analyzed its sub-cellular localization. Using immunostaining as well as gfp reporter assay, we have shown a nuclear localization for PME-5. Moreover, we showed that PME-5 is a ubiquitous nuclear protein expressed throughout the development of the worm and is closely linked to chromatin and condensed chromosomes. Taken together, our data suggest that C. elegans can be used to study the nuclear roles of tankyrase. PMID:19104912

White, Charles; Gagnon, Steve N; St-Laurent, Jean-François; Gravel, Catherine; Proulx, Léa-Isabelle; Desnoyers, Serge



Recruitment of human cyclin T1 to nuclear bodies through direct interaction with the PML protein  

PubMed Central

Human cyclin T1, the cyclin partner of Cdk9 kinase in the positive transcription elongation factor b (P-TEFb), is an essential cellular cofactor that is recruited by the human immunodeficiency virus type 1 (HIV-1) Tat transactivator to promote transcriptional elongation from the HIV-1 long terminal repeat (LTR). Here we exploit fluorescence resonance energy transfer (FRET) to demonstrate that cyclin T1 physically interacts in vivo with the promyelocytic leukaemia (PML) protein within specific subnuclear compartments that are coincident with PML nuclear bodies. Deletion mutants at the C-terminal region of cyclin T1 are negative for FRET with PML and fail to localize to nuclear bodies. Cyclin T1 and PML are also found associated outside of nuclear bodies, and both proteins are present at the chromatinized HIV-1 LTR promoter upon Tat transactivation. Taken together these results suggest that PML proteins regulate Tat- mediated transcriptional activation by modulating the availability of cyclin T1 and other essential cofactors to the transcription machinery.

Marcello, Alessandro; Ferrari, Aldo; Pellegrini, Vittorio; Pegoraro, Gianluca; Lusic, Marina; Beltram, Fabio; Giacca, Mauro



During its nuclear phase the multifunctional regulatory protein ICP0 undergoes proteolytic cleavage characteristic of polyproteins.  


ICP0 is a multifunctional herpes simplex virus protein known primarily as a promiscuous transactivator. In the course of productive infection, it is localized during the first 5-7 h in the nucleus and later in the cytoplasm. In the nucleus, its primary activities are to suppress the silencing of viral DNA by host proteins, activate cdk4 through recruitment of cyclin D3 to the sites of formation of replication compartments, and degrade several cellular proteins including PML and Sp100, key components of the ND10 nuclear bodies. ICP0 is not translocated to the cytoplasm in cells infected with mutants incapable of performing these tasks. We report the unexpected finding that ICP0 is cleaved into several discrete polypeptides by a proteasome-independent process. The products of this cleavage accumulate in cells infected with ICP0 mutants incapable of degrading PML and therefore are retained in the nucleus. In the second step, the products of the initial cleavage of wild-type virus-infected cells are themselves subject to proteasome-dependent degradation. The average half life of intact ICP0 during the nuclear phase is approximately 1 h. The proteasome-independent cleavage products are no longer detected at late times corresponding to the cytoplasmic phase of ICP0. The results are consistent with the hypothesis that the cleavage products of ICP0 function in topologically distinct domains during its nuclear phase. PMID:19850872

Gu, Haidong; Poon, Alice P; Roizman, Bernard



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:; 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)



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

PubMed Central

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 of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes.

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



Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata.  


The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus. PMID:12175672

Holzinger, Andreas; Lütz-Meindl, Ursula



Nucleocytoplasmic shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to promyelocytic leukemia bodies.  


p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84. PMID:20018885

Pankiv, Serhiy; Lamark, Trond; Bruun, Jack-Ansgar; Øvervatn, Aud; Bjørkøy, Geir; Johansen, Terje



A Visual Screen of a Gfp-Fusion Library Identifies a New Type of Nuclear Envelope Membrane Protein  

PubMed Central

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

Rolls, Melissa M.; Stein, Pascal A.; Taylor, Stephen S.; Ha, Edward; McKeon, Frank; Rapoport, Tom A.



The lactate dehydrogenase-elevating virus capsid protein is a nuclear–cytoplasmic protein  

Microsoft Academic Search

Arteriviruses replicate in the cytoplasm and do not require the nucleus function for virus multiplication in vitro. However,\\u000a nucleocapsid (N) protein of two arteriviruses, porcine reproductive respiratory syndrome virus and equine arteritis virus,\\u000a has been observed to localize in the nucleus and nucleolus of virus-infected and N-gene-transfected cells in addition to their\\u000a normal cytoplasmic distribution. In the present study, the

Hakimeh Mohammadi; Shayan Sharif; Raymond R. Rowland; Dongwan Yoo



Nuclear Entry of Hepatitis B Virus Capsids Involves Disintegration to Protein Dimers followed by Nuclear Reassociation to Capsids  

PubMed Central

Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly.

Bischof, Andreas; Foss, Michael; Sominskaya, Irina; Pumpens, Paul; Cazenave, Christian; Castroviejo, Michel; Kann, Michael



The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import.  


The nucleocytoplasmic export of cytomegaloviral capsids is regulated by formation of a multi-component nuclear egress complex (NEC), essentially based on viral proteins pUL50 and pUL53. In this study, the generation of recombinant human cytomegaloviruses, expressing tagged versions of pUL50 and pUL53, enabled the investigation of NEC formation in infected primary fibroblasts. For these recombinant viruses, a wild-type-like mode of pUL50-pUL53 interaction and recruitment of both proteins to the nuclear envelope could be demonstrated. Importantly, pUL50 was translocated from an initial cytoplasmic distribution to the nuclear rim, whereas pUL53 accumulated in the nucleus before attaining overall rim colocalization with pUL50. Specified experimental settings illustrated that pUL50 and pUL53 were subject to different pathways of intracellular trafficking. Importantly, a novel nuclear localization signal (NLS) could be identified and functionally verified for pUL53 (amino acids 18-27), whereas no NLS was present in pUL50. Analysis of amino acid replacement mutants further illustrated the differential modes of nuclear import of the two essential viral egress proteins. Taken together, our findings suggest a combination of classical nuclear import (pUL53) and interaction-mediated recruitment (pUL50) as the driving forces for core NEC formation and viral nuclear egress. PMID:23740483

Schmeiser, Cathrin; Borst, Eva; Sticht, Heinrich; Marschall, Manfred; Milbradt, Jens



Conserved Sr Protein Kinase Functions in Nuclear Import and Its Action Is Counteracted by Arginine Methylation in Saccharomyces cerevisiae  

Microsoft Academic Search

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to en- ter the nucleus through a

Chi Y. Yun; Xiang-Dong Fu



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

PubMed Central

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

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



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


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

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



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

SciTech Connect

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.

Lipsius, Edgar [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Walter, Korden [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Leicher, Torsten [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Phlippen, Wolfgang [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Bisotti, Marc-Angelo [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany); Kruppa, Joachim [Zentrum fuer Experimentelle Medizin, Institut fuer Molekulare Zellbiologie, Universitaet Hamburg, Hamburg (Germany)]. E-mail:



Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins  

PubMed Central

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.



Kinase Activity-dependent Nuclear Export Opposes Stress-induced Nuclear Accumulation and Retention of Hog1 Mitogen-activated Protein Kinase in the Budding Yeast Saccharomyces cerevisiae  

PubMed Central

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1–green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1–GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1–GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Reiser, Vladimir; Ruis, Helmut; Ammerer, Gustav



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


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



Analysis of influenza B Virus NS1 protein trafficking reveals a novel interaction with nuclear speckle domains.  


Many proteins that function in the transcription, maturation, and export of metazoan mRNAs are concentrated in nuclear speckle domains, indicating that the compartment is important for gene expression. Here, we show that the NS1 protein of influenza B virus (B/NS1) accumulates in nuclear speckles and causes rounding and morphological changes of the domains, indicating a disturbance in their normal functions. This property was located within the N-terminal 90 amino acids of the B/NS1 protein and was shown to be independent of any other viral gene product. Within this protein domain, we identified a monopartite importin alpha binding nuclear localization signal. Reverse-genetic analysis of this motif indicated that nuclear import and speckle association of the B/NS1 protein are required for the full replication capacity of the virus. In the late phase of virus infection, the B/NS1 protein relocated to the cytoplasm, which occurred in a CRM1-independent manner. The interaction of the B/NS1 protein with nuclear speckles may reflect a recruitment function to promote viral-gene expression. To our knowledge, this is the first functional description of a speckle-associated protein that is encoded by a negative-strand RNA virus. PMID:18987144

Schneider, Jana; Dauber, Bianca; Melén, Krister; Julkunen, Ilkka; Wolff, Thorsten



Nuclear Import of UBL-Domain Protein Mdy2 Is Required for Heat-Induced Stress Response in Saccharomyces cerevisiae  

PubMed Central

Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-?NES, but not Mdy2-?NLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress.

Arhzaouy, Khalid; Ramezani-Rad, Massoud



Interactions and three-dimensional localization of a group of nuclear pore complex proteins  

PubMed Central

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N- acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold- conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.



Age-specific nuclear proteins in the nematode worm Caenorhabditis elegans.  

PubMed Central

The nematode worm Caenorhabditis elegans is known to undergo characteristic morphological as well as physiological signs of senescence. Two-dimensional gel electrophoresis shows that alterations also occur in the pattern of the nuclear proteins as a function of age. Non-histone proteins whose level exhibits a steep fall with age are egg-specific and not involved in senescence. However, a distinct set of non-histones accumulates with age and can be considered as senescence markers. Some of these are glycoproteins, as shown by their concanavalin A-binding properties. One age-specific polypeptide, called 'protein S-28', was further characterized by peptide mapping and determination of its N-terminal amino acid sequence. Images Fig. 1. Fig. 2. Fig. 3.

Meheus, L A; Van Beeumen, J J; Coomans, A V; Vanfleteren, J R



Modulation of CREB Binding Protein Function by the Promyelocytic (PML) Oncoprotein Suggests a Role for Nuclear Bodies in Hormone Signaling  

Microsoft Academic Search

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. Here, we demonstrate that the cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate

Vassilis Doucas; Marc Tini; David A. Egan; Ronald M. Evans



The PHD Finger of the Chromatin-Associated Protein ING2 Functions as a Nuclear Phosphoinositide Receptor  

Microsoft Academic Search

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that

Gozani; Philip Karuman; David R. Jones; Dmitri Ivanov; James Cha; Alexey A. Lugovskoy; Cheryl L. Baird; Hong Zhu; Seth J. Field; Stephen L. Lessnick; Jennifer Villasenor; Bharat Mehrotra; Jian Chen; Vikram R. Rao; Joan S. Brugge; Colin G. Ferguson; Bernard Payrastre; David G. Myszka; Lewis C. Cantley; Gerhard Wagner; Nullin Divecha; Glenn D. Prestwich; Junying Yuan



Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.  

PubMed Central

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24-nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site-dependent manner. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 6.

Leegwater, P A; van der Vliet, P C; Rupp, R A; Nowock, J; Sippel, A E



The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body–P body pathway  

Microsoft Academic Search

Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, we show that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and

Lin Lee; Siân E. Davies; Ji-Long Liu



Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2.  


A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein. PMID:1333132

Chang, D; Haynes, J I; Brady, J N; Consigli, R A



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.

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



Characterization of the Chaetoceros salsugineum nuclear inclusion virus coat protein gene.  


Analysis of the genome of Chaetoceros salsugineum nuclear inclusion virus (CsNIV) revealed the presence of six putative open reading frames (ORFs) in the genome. We further characterized ORF3, which encodes a putative coat protein. Polymerase chain reaction (PCR) using ORF3 gene-specific primers amplified a single DNA band nearly 1.2kb. This amplified product was gel-purified, cloned, sequenced, and expressed in Escherichia coli. Specific antiserum was raised against the recombinant protein and used for Western blotting to test whether the ORF3 protein is the CsNIV coat protein. One major CsNIV protein of approximately 46kDa reacted positively with the antiserum, suggesting that this antiserum is specific for the CsNIV coat protein. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the 46kDa structural band revealed 14 peptide sequences that matched the ORF3 regions of CsNIV. The expression of ORF3 in host cells was examined by constructing a cDNA library of CsNIV-infected cells. Nucleotide sequences of the cDNA clones were complementary to various regions of both CsNIV ORF3 and ORF4; however, no clones containing only the ORF3 region were identified. Also, Northern blotting revealed a single 2.5-kb transcript, indicating that ORF3 could be transcribed together with ORF4. PMID:19428745

Park, Yunjung; Jung, Sang-Eun; Tomaru, Yuji; Choi, Woobong; Kim, Yoonhee; Mizumoto, Hiroyuki; Nagasaki, Keizo; Choi, Tae-Jin



Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples.  


Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers. PMID:19733545

Ribeiro, João P; Palczewska, Ma?gorzata; André, Sabine; Cañada, F Javier; Gabius, Hans-Joachim; Jiménez-Barbero, Jesús; Mellström, Britt; Naranjo, José R; Scheffers, Dirk-Jan; Groves, Patrick



Deuterium nuclear magnetic resonance study of protein dynamics and protein-lipid interactions in model and biological membranes  

SciTech Connect

The dynamics of the Halobacterium halobium purple membrane protein, bacteriorhodopsin; and the motional order of the fluorescent lipid probe, diphenylhexatriene, in various model membrane systems, have been investigated by deuterium nuclear magnetic resonance (NMR) spectroscopy. /sup 2/H NMR spectra and spin-lattice relaxation times of selectively-deuterated amino acids in the crystalline solid state and in biosynthetically enriched bacteriorhodopsin, have been obtained. Analysis of the data has led to the determination of the type and rate of amino acid motion in these systems. The motions of amino acid residues, are shown to be very sensitive to packing considerations. Large differences in both the type and rate of motion are observed for a particular amino acid in various crystal lattice forms and in the membrane protein. Moreover, motional heterogeneity is found to occur in some crystal lattice forms and especially in bacteriorhodopsin. The major types of motion that are observed include methyl group rotation (by three-fold hops), phenyl ring flips (180/sup 0/ hops), ring libration, chain libration, side-chain hops, and isotropic reorientation. The last two motions are seen only in the membrane protein. All of these motions can occur at widely different rates depending on the packing environment, but in bacteriorhodopsin, these rates are generally greater than 10/sup 6/ sec/sup -1/. /sup 2/H NMR spectra of selectively-deuterated diphenylhexatriene in dimyristoylphosphatidylcholine multilamellar bilayers in the liquid crystalline phase, as a function of temperature and membrane composition, have also been obtained.

Kintanar, A.B.



Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein  

SciTech Connect

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.

Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.



Centrin 2 localizes to the vertebrate nuclear pore and plays a role in mRNA and protein export.  


Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N- or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore localization, and plays a functional role in multiple nuclear export pathways. PMID:18172010

Resendes, Karen K; Rasala, Beth A; Forbes, Douglass J



The inner nuclear membrane protein Emerin regulates ?-catenin activity by restricting its accumulation in the nucleus  

PubMed Central

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of ?-catenin, an important transcription coactivator, into the nucleus. Emerin interacts with ?-catenin through a conserved adenomatous polyposis coli (APC)-like domain. When GFP-emerin is expressed in HEK293 cells, ?-catenin is restricted to the cytoplasm and ?-catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC-like domain (GFP-emerin?), dominantly stimulates ?-catenin activity and increases nuclear accumulation of ?-catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of ?-catenin and can be replicated in wild-type fibroblasts by transfection with constitutively active ?-catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.

Markiewicz, Ewa; Tilgner, Katarzyna; Barker, Nick; van de Wetering, Mark; Clevers, Hans; Dorobek, Margareth; Hausmanowa-Petrusewicz, Irena; Ramaekers, Frans C S; Broers, Jos L V; Blankesteijn, W Matthijs; Salpingidou, Georgia; Wilson, Robert G; Ellis, Juliet A; Hutchison, Christopher J



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

PubMed Central

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

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



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

PubMed Central

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

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



Mammalian Meiotic Telomeres: Protein Composition and Redistribution in Relation to Nuclear Pores  

PubMed Central

Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.

Scherthan, Harry; Jerratsch, Martin; Li, Bibo; Smith, Susan; Hulten, Maj; Lock, Tycho; de Lange, Titia



Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication  

Microsoft Academic Search

Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analy- sis of nuclear transport. Such cell-free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuclear pore. Here, the role of p97 has been investigated. Xenopus p97 was isolated and antisera were

Maureen A. Powers; Colin Macaulay; Frank R. Masiarz; Douglass J. Forbes



Conservation of a Masked Nuclear Export Activity of La Proteins and Its Effects on tRNA Maturation?  

PubMed Central

La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa) can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional processing of associated pre-tRNAs that are spliced but 5? and 3? unprocessed, with an accompanying decrease in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype. NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the multifunctional La protein and that their interrelationship contributes to the ability of La to engage its different classes of RNA ligands in different cellular locations.

Bayfield, Mark A.; Kaiser, Trish E.; Intine, Robert V.; Maraia, Richard J.



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



Interaction of DNA/nuclear protein/polycation and the terplexes for gene delivery.  


Nuclear transport of exogenous DNA is a major barrier to nonviral gene delivery that needs to be addressed in the design of new vectors. In this study, we prepared pDNA/HMGB1/PEG-PEI terplexes to promote nuclear import. HMGB1 in the terplexes was used to assist the transportation of pDNA into the nucleus of cells, since it contained nuclear localization signal (NLS); PEG chains were introduced to stabilize pDNA/vector terplexes and reduce the cytotoxicity. HMGB1/PEG-PEI combined vectors have been investigated specifically for their structure interaction by atomic force microscopy and circular dichroic spectroscopy. The results demonstrated that the HMGB1 molecule could bind with the pDNA chains, but not condense pDNA well. The PEG-PEI further compacted pDNA/HMGB1 complexes into nanosized spherical terplexes. The pDNA delivered by HMGB1/PEG-PEI combined vectors was significantly accumulated in the nucleus of cells, as observed by confocal laser scanning microscopy. The percentage of GFP-transfected cells and VEGF protein expression level induced by HMGB1/PEG-PEI were 2.6-4.9-fold and 1.4-2.8-fold higher, respectively, than that of a common cationic polymer PEI 25 kDa. Therefore, the HMGB1/PEG-PEI combined vector could be used as a versatile vector for promoting exogenous DNA nuclear localization, thereby enhancing its expression. PMID:20009166

Shen, Yuan; Peng, Hui; Pan, Shirong; Feng, Min; Wen, Yuting; Deng, Jingjing; Luo, Xin; Wu, Chuanbin



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

SciTech Connect

We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap {alpha}{sub 2} adapter, and Kap {beta}{sub 2} and Kap {beta}{sub 3} nuclear import receptors. Moreover, binding of RanGTP to either Kap {beta}{sub 2} or Kap {beta}{sub 3} inhibits their interaction with L2, suggesting that these Kap {beta}/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap {alpha}{sub 2}{beta}{sub 1} heterodimer and mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap {beta}{sub 2} and Kap {beta}{sub 3}. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap {alpha}{sub 2}{beta}{sub 1} heterodimers, Kap {beta}{sub 2} and Kap {beta}{sub 3}.

Klucevsek, K. [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Daley, J. [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Darshan, M.S. [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Bordeaux, J. [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Moroianu, J. [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States)]. E-mail:



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

PubMed Central

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



Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin  

PubMed Central

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP–Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.

Fenn, Sebastian; Breitsprecher, Dennis; Gerhold, Christian B; Witte, Gregor; Faix, Jan; Hopfner, Karl-Peter



Synthesis of sperm-specific basic nuclear proteins (SPs) in cultured spermatids from Xenopus laevis  

SciTech Connect

The accumulation and synthesis of sperm-specific basic nuclear proteins (SPs) in Xenopus spermatids in vitro were studied by acid-urea-Triton polyacrylamide gel electrophoresis and fluorography. In synchronous cultures of round spermatids, the amount of SP2 and SP3-5 accumulated almost linearly with time, while that of SP1 remained almost constant. Fluorography showed that round spermatids incorporated {sup 14}C arginine mostly into SP1 and SP3-5, very little into SP2, and none into histones. When {sup 14}C arginine was incorporated into cells for 24 h on Days 0, 3, and 6, followed by immediate extraction of basic nuclear proteins, the SP1 band was detected faintly on Day 0 and the intensity increased to the maximum level by Day 3 and remained constant on Day 6; the SP3-5 bands were first detected on Day 3 and their intensity increased by Day 6. Thus, SP1 and SP3-5 were synthesized differentially during the culture period. When {sup 14}C arginine or {sup 14}C lysine was incorporated into round spermatids on Days 0, 3, and 6 for 15 h and chased for 3-12 days, the intensity of the SP2 band increased significantly, while the intensity of the SP1 band decreased concomitantly. This result indicates that SP2 was processed from a precursor protein which is probably SP1.

Abe, S.; Hiyoshi, H. (Kumamoto Univ. (Japan))



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:



Identification of a Heterogeneous Nuclear Ribonucleoprotein-recognition Region in the HIV Rev Protein*  

PubMed Central

The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9–14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions.

Hadian, Kamyar; Vincendeau, Michelle; Mausbacher, Nina; Nagel, Daniel; Hauck, Stefanie M.; Ueffing, Marius; Loyter, Abraham; Werner, Thomas; Wolff, Horst; Brack-Werner, Ruth



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


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. PMID:21489587

Salsman, Jayme; Wang, Xueqi; Frappier, Lori



Emerging Roles for the Influenza A Virus Nuclear Export Protein (NEP)  

PubMed Central

Influenza virus is a major human and animal pathogen causing seasonal epidemics and occasional pandemics in the human population that are associated with significant morbidity and mortality. Influenza A virus, a member of the orthomyxovirus family, contains an RNA genome with a coding capacity for a limited number of proteins. In addition to ensuring the structural integrity of virions, these viral proteins facilitate the replication of virus in the host cell. Consequently, viral proteins often evolve to perform multiple functions, the influenza A virus nuclear export protein (NEP) (also referred to as non-structural protein 2, or NS2) being an emerging example. NEP was originally implicated in mediating the nuclear export of viral ribonucleoprotein (RNP) complexes, which are synthesized in the infected cell nucleus and are assembled into progeny virions at the cell membrane. However, since then, new and unexpected roles for NEP during the influenza virus life cycle have started to emerge. These recent studies have shown NEP to be involved in regulating the accumulation of viral genomic vRNA and antigenomic cRNA as well as viral mRNA synthesized by the viral RNA-dependent RNA polymerase. Subsequently, this regulation of viral RNA transcription and replication by NEP was shown to be an important factor in the adaptation of highly pathogenic avian H5N1 influenza viruses to the mammalian host. Unexpectedly, NEP has also been implicated in recruiting a cellular ATPase to the cell membrane to aid the efficient release of budding virions. Accordingly, NEP is proposed to play multiple biologically important roles during the influenza virus life cycle.

Paterson, Duncan; Fodor, Ervin



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:



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


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

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



Prediction of leucine-rich nuclear export signal containing proteins with NESsential  

PubMed Central

The classical nuclear export signal (NES), also known as the leucine-rich NES, is a protein localization signal often involved in important processes such as signal transduction and cell cycle regulation. Although 15 years has passed since its discovery, limited structural information and high sequence diversity have hampered understanding of the NES. Several consensus sequences have been proposed to describe it, but they suffer from poor predictive power. On the other hand, the NetNES server provides the only computational method currently available. Although these two methods have been widely used to attempt to find the correct NES position within potential NES-containing proteins, their performance has not yet been evaluated on the basic task of identifying NES-containing proteins. We propose a new predictor, NESsential, which uses sequence derived meta-features, such as predicted disorder and solvent accessibility, in addition to primary sequence. We demonstrate that it can identify promising NES-containing candidate proteins (albeit at low coverage), but other methods cannot. We also quantitatively demonstrate that predicted disorder is a useful feature for prediction and investigate the different features of (predicted) ordered versus disordered NES’s. Finally, we list 70 recently discovered NES-containing proteins, doubling the number available to the community.

Fu, Szu-Chin; Imai, Kenichiro; Horton, Paul



Sperm nuclear basic proteins of tunicates and the origin of protamines.  


Sperm nuclear basic proteins (SNBPs) are the chromosomal proteins that are found associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins can be classified into three major types: histone type (H-type), protamine-like type (PL-type), and protamine type (P-type). A hypothesis from early studies on the characterization of SNBPs proposed a mechanism for the vertical evolution of these proteins that involved an H1 ? PL ? P transition. However, the processes and mechanisms involved in such a transition were not understood. In particular, it was not clear how a molecular transition from a lysine-rich protein precursor (H1 histone) to the arginine-rich protamines might have taken place. In deuterostomes, the presence of SNBPs of the H-type in echinoderms and of protamines in the higher phylogenetic groups of vertebrates had long been known. The initial work on the characterization of tunicate SNBPs attempted to define the types and range of SNBPs that characterize this phylogenetically intermediate group. It was found that tunicate SNBPs belong to the PL-type. In this work we discuss how the study of SNBPs in the tunicates has been key to providing support to the H1 ? PL ? P transition. Most significantly, it was in tunicates that a potential molecular mechanism to explain the lysine-to-arginine transition was first reported. PMID:23995738

Saperas, Núria; Ausió, Juan



Preferential modification of nuclear proteins by a novel ubiquitin-like molecule.  


Sentrin is a novel ubiquitin-like protein that protects cells against both anti-Fas and tumor necrosis factor-induced cell death. Antiserum recognizing the N terminus of sentrin revealed the presence of a 18-kDa sentrin monomer, a 90-kDa band (p90), and multiple high molecular mass bands. Because sentrin possesses the conserved Gly-Gly residues near the C terminus, it is likely that these additional bands represent conjugation of sentrin to other proteins in a manner that is similar to the ubiquitination pathway. Transient expression of hemagglutinin epitope-tagged sentrin mutants in COS cells demonstrated that the sentrin C terminus is cleaved, which allows it to be conjugated to other proteins via the conserved C-terminal Gly residue. Immunocytochemical staining and cell fractionation analysis demonstrated that sentrin monomer is localized predominantly to the cytosol. However, p90 and the majority of sentrinized proteins appeared to be localized to the nucleus. When the conserved Gly-Gly residues of sentrin were changed to Gly-Ala, only sentrin monomer and p90 but not the high molecular mass bands were observed. Thus, p90 generation appears to be required for the formation of high molecular mass bands in the nucleus. Taken together, sentrinization represents a novel pathway for nuclear protein modification, which is distinct from ubiquitination. PMID:9162015

Kamitani, T; Nguyen, H P; Yeh, E T



Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes  

PubMed Central

The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ?50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting.

Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Payne, H. Ross; Kier, Ann B.



Non-canonical function of Bax in stress-induced nuclear protein redistribution.  


Bax and Bak (Bax/Bak) are essential pro-apoptotic proteins of the Bcl-2 family that trigger mitochondrial outer membrane permeabilization (MOMP) in a Bcl-2/Bcl-xL-inhibitable manner. We recently discovered a new stress-related function for Bax/Bak-regulation of nuclear protein redistribution (NPR) from the nucleus to cytoplasm. This effect was independent of Bax/Bak N-terminus exposure and not inhibited by Bcl-xL over-expression. Here, we studied the molecular mechanism governing this novel non-canonical response. Wild-type (WT) and mutant versions of Bax were re-expressed in Bax/Bak double-knockout mouse embryonic fibroblasts and their ability to promote NPR, apoptotic events, and changes in lamin A mobility was examined. Our results show that, in this system, Bax expression was sufficient to restore NPR such as in WT cells undergoing apoptosis. This activity of Bax was uncoupled from cytochrome c release from the mitochondria (indicative of MOMP) and required its membrane localization, ? helices 5/6, and the Bcl-2 homology 3 (BH3) domain. Moreover, enrichment of Bax in the nuclear envelope by the so-called Klarsicht/ANC-1/Syne-1 homology domain effectively triggered NPR as in WT Bax, but without inducing MOMP or cell death. Bax-induced NPR was associated with impairment in lamin A mobility, implying a connection between these two nuclear envelope-associated events. Overall, the results indicate a new MOMP-independent, stress-induced Bax function on the nuclear envelope. PMID:23475110

Lindenboim, Liora; Ferrando-May, Elisa; Borner, Christoph; Stein, Reuven



Importin beta-depending nuclear import pathways: role of the adapter proteins in the docking and releasing steps.  


Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin beta. However, due to the presence of different import signals, the adapter protein of the imported molecules and importin beta is different for each pathway. Although the adapter for cNLS-protein is importin alpha, the adapter for U1 snRNP is snurportin1 (SPN1). Herein, we show that the use of distinct adapters by importin beta results in differences at the docking and releasing step for these two import pathways. Nuclear pore complex (NPC) docking of U1 snRNP but not of cNLS-protein was inhibited by an anti-CAN/Nup214 antibody. Thus, the initial NPC-binding site is different for each pathway. Pull-down assays between immobilized SPN1 and two truncated forms of importin beta documented that SPN1 and importin alpha have different binding sites on importin beta. Importin beta fragment 1-618, which binds to SPN1 but not to importin alpha, was able to support the nuclear import of U1 snRNPs. After the translocation through the NPC, both import complexes associated with the nuclear side of the NPC. However, we found that the nature of the importin beta-binding domain of the adapters influences the release of the cargo into the nucleoplasm. PMID:12802078

Rollenhagen, Christiane; Mühlhäusser, Petra; Kutay, Ulrike; Panté, Nelly



Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation  

Microsoft Academic Search

BACKGROUND: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue

Beate Reichart; Ruth Klafke; Christine Dreger; Eleonora Krüger; Isabell Motsch; Andrea Ewald; Jochen Schäfer; Heinz Reichmann; Clemens R Müller; Marie-Christine Dabauvalle



An N-terminal nuclear export signal regulates trafficking and aggregation of Huntingtin (Htt) protein exon 1.  


Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity. PMID:23319588

Zheng, Zhiqiang; Li, Aimin; Holmes, Brandon B; Marasa, Jayne C; Diamond, Marc I



The kinesin-like protein TOP promotes Aurora localisation and induces mitochondrial, chloroplast and nuclear division.  


The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear, mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae. A proteomics study revealed that TOP is a member of a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original function of C-terminal kinesin-like proteins. PMID:23549784

Yoshida, Yamato; Fujiwara, Takayuki; Imoto, Yuuta; Yoshida, Masaki; Ohnuma, Mio; Hirooka, Shunsuke; Misumi, Osami; Kuroiwa, Haruko; Kato, Shoichi; Matsunaga, Sachihiro; Kuroiwa, Tsuneyoshi



Electron transfer mechanism of the Rieske protein from Thermus thermophilus from solution nuclear magnetic resonance investigations.  


We report nuclear magnetic resonance (NMR) data indicating that the Rieske protein from the cytochrome bc complex of Thermus thermophilus (TtRp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. To test models concerning the mechanism by which TtRp transfers between different sites on the complex, we monitored (1)H, (15)N, and (13)C NMR signals as a function of the redox state and molar ratio of added ligand. Our studies of full-length TtRp were conducted in the presence of dodecyl phosphocholine micelles to solvate the membrane anchor of the protein and the hydrophobic tail of the ligand (hydroubiquinone). NMR data indicated that hydroubiquinone binds to TtRp and stabilizes an altered protein conformation. We utilized a truncated form of the Rieske protein lacking the membrane anchor (trunc-TtRp) to investigate redox-state-dependent conformational changes. Local chemical shift perturbations suggested possible conformational changes at prolyl residues. Detailed investigations showed that all observable prolyl residues of oxidized trunc-TtRp have trans peptide bond configurations but that two of these peptide bonds (Cys151-Pro152 and Gly169-Pro170 located near the iron-sulfur cluster) become cis in the reduced protein. Changes in the chemical shifts of backbone signals provided evidence of redox-state- and ligand-dependent conformational changes localized near the iron-sulfur cluster. These structural changes may alter interactions between the Rieske protein and the cytochrome b and c sites and provide part of the driving force for movement of the Rieske protein between these two sites. PMID:23480240

Hsueh, Kuang-Lung; Tonelli, Marco; Cai, Kai; Westler, William M; Markley, John L



The Arabidopsis chloroplast ribosomal protein L21 is encoded by a nuclear gene of mitochondrial origin.  


Many chloroplast genes of cyanobacterial origin have been transferred to the nucleus during evolution and their products are re-addressed to chloroplasts. The RPL21 gene encoding the plastid r-protein L21 has been lost in higher plant chloroplast genomes after the divergence from bryophytes. Based on phylogenetic analysis and intron conservation, we now provide evidence that in Arabidopsis a nuclear RPL21c gene of mitochondrial origin has replaced the chloroplast gene. The control of expression of this gene has been adapted to the needs of chloroplast development by the acquisition of plastid-specific regulatory promoter cis-elements. PMID:11675010

Gallois, J L; Achard, P; Green, G; Mache, R



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


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, its biological activities and its pathological effects in sepsis, arthritis, cancer and other diseases. Here, we discuss these features of HMGB1 and summarize recent advances that have led to the preclinical development of therapeutics that modulate HMGB1 release and activity. PMID:15803152

Lotze, Michael T; Tracey, Kevin J



Mechanism of activation of NDR (nuclear Dbf2-related) protein kinase by the hMOB1 protein.  


NDR (nuclear Dbf2-related) kinase belongs to a family of kinases that is highly conserved throughout the eukaryotic world. We showed previously that NDR is regulated by phosphorylation and by the Ca(2+)-binding protein, S100B. The budding yeast relatives of Homo sapiens NDR, Cbk1, and Dbf2, were shown to interact with Mob2 (Mps one binder 2) and Mob1, respectively. This interaction is required for the activity and biological function of these kinases. In this study, we show that hMOB1, the closest relative of yeast Mob1 and Mob2, stimulates NDR kinase activity and interacts with NDR both in vivo and in vitro. The point mutations of highly conserved residues within the N-terminal domain of NDR reduced NDR kinase activity as well as human MOB1 binding. A novel feature of NDR kinases is an insert within the catalytic domain between subdomains VII and VIII. The amino acid sequence within this insert shows a high basic amino acid content in all of the kinases of the NDR family known to interact with MOB proteins. We show that this sequence is autoinhibitory, and our data indicate that the binding of human MOB1 to the N-terminal domain of NDR induces the release of this autoinhibition. PMID:15197186

Bichsel, Samuel J; Tamaskovic, Rastislav; Stegert, Mario R; Hemmings, Brian A



Anchorage of plant RanGAP to the nuclear envelope involves novel nuclear-pore-associated proteins.  


The Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, and nuclear envelope (NE) formation [1-4]. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1 [5-8]. Mammalian RanGAP1 targeting to the NE and kinetochores requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2 [9-14]. In contrast, Arabidopsis RanGAP1 is associated with the NE and cell plate, mediated by an N-terminal, plant-specific WPP domain [15-18]. In the absence of RanBP2 in plants, the mechanism for spatially sequestering plant RanGAP is unknown. Here, Arabidopsis WPP-domain interacting proteins (WIPs) that interact with RanGAP1 in vivo and colocalize with RanGAP1 at the NE and cell plate were identified. Immunogold labeling indicates that WIP1 is associated with the outer NE. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root-tip cells, whereas NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIPs are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms. PMID:17600715

Xu, Xianfeng Morgan; Meulia, Tea; Meier, Iris



Targeting Promyelocytic Leukemia Protein: A Means to Regulating PML Nuclear Bodies  

PubMed Central

The promyelocytic leukemia protein (PML) is involved in many cellular processes including cell cycle progression, DNA damage response, transcriptional regulation, viral infection, and apoptosis. These cellular activities often rely on the localization of PML to unique subnuclear structures known as PML nuclear bodies (NBs). More than 50 cellular proteins are known to traffic in and out of PML NBs, either transiently or constitutively. In order to understand the dynamics of these NBs, it is important to delineate the regulation of PML itself. PML is subject to extensive regulation at transcriptional, post-transcriptional, and post-translational levels. Many of these modes of regulation depend on the cellular context and the presence of extracellular signals. This review focuses on the current knowledge of regulation of PML under normal cellular conditions as well as the role for regulation of PML in viral infection and cancer.

Reineke, Erin L.; Kao, Hung-Ying



Assessment of higher order structure comparability in therapeutic proteins using nuclear magnetic resonance spectroscopy.  


In this work, we applied nuclear magnetic resonance (NMR) spectroscopy to rapidly assess higher order structure (HOS) comparability in protein samples. Using a variation of the NMR fingerprinting approach described by Panjwani et al. [2010. J Pharm Sci 99(8):3334-3342], three nonglycosylated proteins spanning a molecular weight range of 6.5-67?kDa were analyzed. A simple statistical method termed easy comparability of HOS by NMR (ECHOS-NMR) was developed. In this method, HOS similarity between two samples is measured via the correlation coefficient derived from linear regression analysis of binned NMR spectra. Applications of this method include HOS comparability assessment during new product development, manufacturing process changes, supplier changes, next-generation products, and the development of biosimilars to name just a few. We foresee ECHOS-NMR becoming a routine technique applied to comparability exercises used to complement data from other analytical techniques. PMID:23568791

Amezcua, Carlos A; Szabo, Christina M



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


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



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.

Redrejo-Rodriguez, Modesto; Munoz-Espin, Daniel; Holguera, Isabel; Mencia, Mario; Salas, Margarita



Distribution of [241Am] in hepato-pancreatic nuclear proteins of the lobster (Homarus gammarus).  


Recent studies on the bio-accumulation of transuranic elements in marine invertebrates have demonstrated an intracellular concentration of radionuclides in the digestive gland. The present investigation aims to establish the distribution of [241Am] within the different structures of the cell nucleus. Lobsters were experimentally contaminated through feeding and then killed. The nuclei of hepato-pancreatic cells were isolated, purified and dissociated. The different protein categories were separated in media of increasing ionic strength. The distribution of americium in this material yielded results showing a preferential fixation onto the structural proteins of the nuclear matrix. The radiological consequences of such an affinity are discussed in terms of the life-times of the ligands concerned. PMID:8490574

Paquet, F; Durand, J P; Goudard, F; Germain, P; Milcent, M C; Pieri, J



Study of nuclear proteins in normal and xeroderma pigmentosum lymphoblastoid cells  

SciTech Connect

Nuclear histone and nonhistone (NHP) proteins from normal human and xeroderma pigmentosum, complementation group A (XP-A) lymphoblastoid cells were compared both qualitatively, quantitatively and for binding affinity for DNA. Histones and four NHP fractions (NHP/sub 1-4/) were isolated from purified cell nuclei. Binding affinity to (/sup 3/H) melanoma DNA of histones and each NHP fraction was then determined using gradient dialysis followed by a filter assay. Histones and each NHP fraction were then sub-fractionated by polyacrylamide gel electrophoresis. Densitometric scans of the separation of these proteins on the gels were qualitatively, and quantitatively analyzed and compared between the two cell lines. No qualitative or quantitative differences were observed between histones from XP-A or normal cells.

Amari, N.M.B.



Cross-Regulation of Protein Stability by p53 and Nuclear Receptor SHP  

PubMed Central

We report here a novel interplay between tumor suppressor p53 and nuclear receptor SHP that controls p53 and SHP stability. Overexpression of p53 causes rapid SHP protein degradation, which does not require the presence of Mdm2 and is mediated by the proteosome pathway. Overexpressing SHP alone does not affect p53 stability. However, SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. The single amino acid substitution in the SHP protein SHPK170R increases SHP binding to p53 relative to SHP wild-type, whereas SHPG171A variant shows a diminished p53 binding. As a result of the cross-regulation, the tumor suppressor function of p53 and SHP in inhibition of colon cancer growth is compromised. Our findings reveal a unique scenario for a cross-inhibition between two tumor suppressors to keep their expression and function in check.

Yang, Zhihong; Zhang, Yuxia; Kemper, Jongsook Kim; Wang, Li



The sequence-specific nuclear matrix binding factor F6 is a chicken GATA-like protein  

Microsoft Academic Search

The sequence-specific DNA-binding protein factor F6, which binds upstream of the cluster of the chicken a-globin genes, has previously been found to interact with a DNA fragment containing a replication origin and a nuclear matrix binding site. This protein has been partially characterized. Based on its molecular weight and binding affinity, F6 belongs to a family of GATA proteins, the

Y. S. Vassetzky; C. V. Moura Gallo; A. N. Bogdanova; S. V. Razin; K. Scherrer



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



The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation  

PubMed Central

Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3?-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing.

Droll, Dorothea; Archer, Stuart; Fenn, Katelyn; Delhi, Praveen; Matthews, Keith; Clayton, Christine



Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins  

PubMed Central

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5? end or 3? end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5? flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5? and 3? ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats.

Galopier, Aurelie; Hermann-Le Denmat, Sylvie



Binding of HeLa cell nuclear proteins to a human chromosome 9 specific repetitive DNA sequence  

SciTech Connect

In order to investigate DNA-protein interactions in defined chromosome regions, the authors have initiated studies to detect the specific binding of proteins from a crude HeLe cell nuclear extract to a cloned human chromosome 9 specific satellite III DNA sequence. Approximately 150,000 copies of this sequence, representing 0.23% of human DNA, are localized in chromosome region 9qh. Using a band retardation assay, in which the migration of a radiolabeled DNA probe is altered by protein binding, two discrete DNA-protein complexes were observed. Kinetic analysis of the formation and dissociation of these two complexes suggests that the slower migrating complex may be a dimer of the faster migrating complex. Competition experiments, in which these specific DNA-protein complexes are formed in the presence of increasing amounts of E. coli, pBr 322, or poly d(I-c) DNA indicate that the HeLa nuclear protein(s) has an approximately 20,000 fold greater affinity for the chromosome 9 specific DNA sequence. Preliminary experiments indicate that the formation of these specific DNA-protein complexes are not blocked by either cloned Alu repetitive DNA sequences, or a related chromosome 16 satellite II repetitive DNA sequence. This suggests that some nuclear proteins may have distinct chromosomal locations.

Grady, D.; Robinson, D.; Moyzis, R.




PubMed Central

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C14-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.

Allfrey, V. G.; Littau, V. C.; Mirsky, A. E.



Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity  

PubMed Central

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.

Carl, Philip L; Temple, Brenda RS; Cohen, Philip L



A novel nuclear structure containing the survival of motor neurons protein.  

PubMed Central

Spinal muscular atrophy (SMA) is a common, often fatal, autosomal recessive disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. A gene termed survival of motor neurons (SMN), at 5q13, has been identified as the determining gene of SMA (Lefebvre et al., 1995). The SMN gene is deleted in > 98% of SMA patients, but the function of the SMN protein is unknown. In searching for hnRNP-interacting proteins we found that SMN interacts with the RGG box region of hnRNP U, with itself, with fibrillarin and with several novel proteins. We have produced monoclonal antibodies to the SMN protein, and we report here on its striking cellular localization pattern. Immunolocalization studies using SMN monoclonal antibodies show several intense dots in HeLa cell nuclei. These structures are similar in number (2-6) and size (0.1-1.0 micron) to coiled bodies, and frequently are found near or associated with coiled bodies. We term these prominent nuclear structures gems, for Gemini of coiled bodies. Images

Liu, Q; Dreyfuss, G



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:



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

Bischoff, D S; Slavicek, J M



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

PubMed Central

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

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



A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.  


Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells. PMID:7813444

Doye, V; Wepf, R; Hurt, E C



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



Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specific DNA-binding protein.  

PubMed Central

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response; the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492-5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP D0B for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250-400 nM, indicating that the protein binds to the CR2 promoter in vivo. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.

Tolnay, M; Vereshchagina, L A; Tsokos, G C



Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.  

PubMed Central

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. Images

Steiner, G; Hartmuth, K; Skriner, K; Maurer-Fogy, I; Sinski, A; Thalmann, E; Hassfeld, W; Barta, A; Smolen, J S



Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function  

PubMed Central

Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.

Byun, Hyewon; Halani, Nimita; Mertz, Jennifer A.; Ali, Almas F.; Lozano, Mary M.; Dudley, Jaquelin P.



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

Bohnlein, E; Gruss, P



The nuclear pore complex protein ALADIN is mislocalized in triple A syndrome  

PubMed Central

Triple A syndrome is a human autosomal recessive disorder characterized by an unusual array of tissue-specific defects. Triple A syndrome arises from mutations in a WD-repeat protein of unknown function called ALADIN (also termed Adracalin or AAAS). We showed previously that ALADIN localizes to nuclear pore complexes (NPCs), large multiprotein assemblies that are the sole sites of nucleocytoplasmic transport. Here, we present evidence indicating that NPC targeting is essential for the function of ALADIN. Characterization of mutant ALADIN proteins from triple A patients revealed a striking effect of these mutations on NPC targeting. A variety of disease-associated missense, nonsense, and frameshift mutations failed to localize to NPCs and were found predominantly in the cytoplasm. Microscopic analysis of cells from a triple A patient revealed no morphological abnormalities of the nuclei, nuclear envelopes, or NPCs. Importantly, these findings indicate that defects in NPC function, rather than structure, give rise to triple A syndrome. We propose that ALADIN plays a cell type-specific role in regulating nucleocytoplasmic transport and that this function is essential for the proper maintenance and/or development of certain tissues. Our findings provide a foundation for understanding the molecular basis of triple A syndrome and may lead to unique insights into the role of nucleocytoplasmic transport in adrenal function and neurodevelopment.

Cronshaw, Janet M.; Matunis, Michael J.



Arabidopsis MDA1, a Nuclear-Encoded Protein, Functions in Chloroplast Development and Abiotic Stress Responses  

PubMed Central

Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis.

Robles, Pedro; Micol, Jose Luis; Quesada, Victor



FHL2 Protein Is a Novel Co-repressor of Nuclear Receptor Nur77*  

PubMed Central

The three members of the NR4A orphan nuclear receptor subfamily Nur77, Nurr1, and NOR-1, regulate a variety of biological functions including vascular disease and metabolism. In this study, we identified Four and a half LIM domains protein-2 (FHL2) as a novel interacting protein of NR4A nuclear receptors by yeast two-hybrid screen and co-immunoprecipitation studies. Each of the four LIM domains of FHL2 can bind Nur77, and both the amino-terminal domain and the DNA binding domain of Nur77 are involved in the interaction between FHL2 and Nur77. FHL2 represses Nur77 transcriptional activity in a dose-dependent manner, and short hairpin RNA-mediated knockdown of FHL2 results in increased Nur77 transcriptional activity. ChIP experiments on the enolase3 promoter revealed that FHL2 inhibits the association of Nur77 with DNA. FHL2 is highly expressed in human endothelial and smooth muscle cells, but not in monocytes or macrophages. To substantiate functional involvement of FHL2 in smooth muscle cell physiology, we demonstrated that FHL2 overexpression increases the growth of these cells, whereas FHL2 knockdown results in reduced DNA synthesis. Collectively, these studies suggest that association of FHL2 with Nur77 plays a pivotal role in vascular disease.

Kurakula, Kondababu; van der Wal, Erik; Geerts, Dirk; van Tiel, Claudia M.; de Vries, Carlie J. M.



The Alphaherpesvirus Serine/Threonine Kinase Us3 Disrupts Promyelocytic Leukemia Protein Nuclear Bodies?†  

PubMed Central

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection, including preventing virus-induced apoptosis, facilitating nuclear egress of capsids, stimulating mRNA translation and promoting cell-to-cell spread of virus infection. Given this diversity, the full spectrum of Us3 function may not yet be recognized. We noted, in transiently transfected cells, that herpes simplex virus type 2 (HSV-2) Us3 disrupted promyelocytic leukemia protein nuclear bodies (PML-NBs). However, PML-NB disruption was not observed in cells expressing catalytically inactive HSV-2 Us3. Analysis of PML-NBs in Vero cells transfected with pseudorabies virus (PRV) Us3 and those in Vero cells infected with Us3-null or -repaired PRV strains indicated that PRV Us3 expression also leads to the disruption of PML-NBs. While loss of PML-NBs in response to Us3 expression was prevented by the proteasome inhibitor MG132, Us3-mediated degradation of PML was not observed in infected cells or in transfected cells expressing enhanced green fluorescent protein (EGFP)-tagged PML isoform IV. These findings demonstrate that Us3 orthologues derived from distantly related alphaherpesviruses cause a disruption of PML-NBs in a kinase- and proteasome-dependent manner but, unlike the alphaherpesvirus ICP0 orthologues, do not target PML for degradation.

Jung, Masany; Finnen, Renee L.; Neron, Casey E.; Banfield, Bruce W.



Nuclear receptors in early hormone refractory prostate cancer and their relationship to apoptosis-related proteins.  


The expression of several genes involved in apoptosis and cell cycle control can be regulated by steroid hormones and related agents via their nuclear receptors. Members of the bcl-2 gene family participate in the regulation of apoptosis in a diverse range of cell types and are implicated in the development of hormone refractory prostate cancer and resistance to anti-cancer therapy. The aim of this study, therefore, was to examine the expression of several nuclear receptors in relation to the expression of apoptosis and cell cycle related proteins in a series of patients with early hormone refractory prostate cancer. Analysis of protein expression revealed only a weak association between Bcl-2 and AR. Bax positivity and p27Kip1 expression were significantly more frequent in the AR-positive tumors, whereas RXRbeta expression was more frequently observed in the AR-negative group. The expression of AR, Bax and p27Kip1 was inversely related, and the expression of RXRbeta directly related, to Gleason pattern status. These results suggest that the immunophenotype of early hormone refractory prostate cancer may be different to that seen in more advanced stage disease. Androgen withdrawal therapy employing anti-androgens may elicit different signalling pathways that may be dependent on ARstatus and ARsensitivity. PMID:12098003

Kolár, Z; Murray, P G; Madarova, J; Lukesova, M; Hlobilkova, A; Riháková, P; Flavell, P; Strnad, M; Student, V; Vojtesek, B



A short Id2 protein fragment containing the nuclear export signal forms amyloid-like fibrils  

SciTech Connect

The negative regulator of DNA-binding/cell-differentiation Id2 is a small protein containing a central helix-loop-helix (HLH) motif and a C-terminal nuclear export signal (NES). Whereas the former is essential for Id2 dimerization and nuclear localization, the latter is responsible for the transport of Id2 from the nucleus to the cytoplasm. Whereas the isolated Id2 HLH motif is highly helical, large C-terminal Id2 fragments including the NES sequence are either unordered or aggregation-prone. To study the conformational properties of the isolated NES region, we synthesized the Id2 segment 103-124. The latter was insoluble in water and only temporarily soluble in water/alcohol mixtures, where it formed quickly precipitating {beta}-sheets. Introduction of a positively charged N-terminal tail prevented aggressive precipitation and led to aggregates consisting of long fibrils that bound thioflavin T. These results show an interesting structural aspect of the Id2 NES region, which might be of significance for both protein folding and function.

Colombo, Noemi [Fakultaet fuer Chemie und Pharmazie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg (Germany); Schroeder, Josef [Institut fuer Pathologie, Zentrales EM-Labor, Fakultaet fuer Medizin, Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg (Germany); Cabrele, Chiara [Fakultaet fuer Chemie und Pharmazie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg (Germany)]. E-mail:



Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.  


The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import. PMID:10356319

Friedler, A; Zakai, N; Karni, O; Friedler, D; Gilon, C; Loyter, A



A Nuclear Matrix Protein Interacts with the Phosphorylated C-Terminal Domain of RNA Polymerase II  

PubMed Central

Yeast two-hybrid screening has led to the identification of a family of proteins that interact with the repetitive C-terminal repeat domain (CTD) of RNA polymerase II (A. Yuryev et al., Proc. Natl. Acad. Sci. USA 93:6975–6980, 1996). In addition to serine/arginine-rich SR motifs, the SCAFs (SR-like CTD-associated factors) contain discrete CTD-interacting domains. In this paper, we show that the CTD-interacting domain of SCAF8 specifically binds CTD molecules phosphorylated on serines 2 and 5 of the consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. In addition, we demonstrate that SCAF8 associates with hyperphosphorylated but not with hypophosphorylated RNA polymerase II in vitro and in vivo. This result suggests that SCAF8 is not present in preinitiation complexes but rather associates with elongating RNA polymerase II. Immunolocalization studies show that SCAF8 is present in granular nuclear foci which correspond to sites of active transcription. We also provide evidence that SCAF8 foci are associated with the nuclear matrix. A fraction of these sites overlap with a subset of larger nuclear speckles containing phosphorylated polymerase II. Taken together, our results indicate a possible role for SCAF8 in linking transcription and pre-mRNA processing.

Patturajan, Meera; Wei, Xiangyun; Berezney, Ronald; Corden, Jeffry L.



The strategy for coupling the RanGTP gradient to nuclear protein export  

PubMed Central

The Ran GTPase plays critical roles in both providing energy for and determining the directionality of nucleocytoplasmic transport. The mechanism that couples the RanGTP gradient to nuclear protein export will determine the rate of and limits to accumulation of export cargoes in the cytoplasm, but is presently unknown. We reasoned that plausible coupling mechanisms could be distinguished by comparing the rates of reverse motion of export cargoes through the nuclear pore complex (NPC) with the predictions of a mathematical model. Measurement of reverse export rates in Xenopus oocytes revealed that nuclear export signals can facilitate RanGTP-dependent cargo movement into the nucleus against the RanGTP gradient at rates comparable to export rates. Although export cargoes with high affinity for their receptor are exported faster than those with low affinity, their reverse transport is also greater. The ratio of the rates of reverse and forward export of a cargo is proportional to its rate of diffusion through the NPC, i.e., to the ability of the cargo to penetrate the NPC permeability barrier. The data substantiate a diffusional mechanism of coupling and suggest the existence of a high concentration of RanGTP-receptor complexes within the NPC that decreases sharply at the cytoplasmic boundary of the NPC permeability barrier.

Becskei, Attila; Mattaj, Iain W.



A conserved CCCH-type zinc finger protein regulates mRNA nuclear adenylation and export.  


Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export. PMID:19364924

Hurt, Jessica A; Obar, Robert A; Zhai, Bo; Farny, Natalie G; Gygi, Steven P; Silver, Pamela A



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

PubMed Central

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




NSDL National Science Digital Library

What part does nuclear energy play in satisfying energy demands? This informational piece, part of a series about the future of energy, introduces students to the uranium atom as an energy source. Here students read about the history of nuclear energy, how energy is derived from uranium, and benefits of nuclear energy. Information is also provided about limitations, particularly disposal problems and radioactivity, and geographical considerations of nuclear power in the United States. Thought-provoking questions afford students chances to reflect on what they've read about the uses of nuclear power. Articles and information on new nuclear plant design and nuclear accidents are available from a sidebar. Five energy-related PBS NewsHour links are provided. A web link to the U.S. Nuclear Regulatory Commission is included. Copyright 2005 Eisenhower National Clearinghouse

Project, Iowa P.



Rhubarb inhibits hepatocellular carcinoma cell metastasis via GSK-3-? activation to enhance protein degradation and attenuate nuclear translocation of ?-catenin.  


The aim of our study was to investigate the mechanisms by which rhubarb regulates ?-catenin as well as metastasis of hepatocellular carcinomas. Our results revealed that rhubarb extract inhibited HA22T cell migration ability in wound healing, migration and invasion assays in a dose-dependent manner. Rhubarb also reduced ?-catenin protein level, downregulated its downstream proteins, cyclin D, Tbx3 and c-Myc, and attenuated the expression of MMP9 and contactin-1 metastatic factors. Additionally, rhubarb inhibited ?-catenin nuclear accumulation and induced its degradation via proteasome-mediated pathway. Furthermore, we found that rhubarb suppressed the p-ser(9) GSK-3-? protein level to inactivate Wnt signalling and reduce ?-catenin protein level. Taken together; we found that rhubarb blocked the metastatic process of HA22T hepatocellular carcinoma cells mediated through GSK-3-? activation, and enhancement of protein degradation as well as reduction of the nuclear accumulation of ?-catenin. PMID:23265488

Tsai, Kun-Hsi; Hsien, Hau-Hsueh; Chen, Li-Mien; Ting, Wei-Jen; Yang, Yuh-Shyong; Kuo, Chia-Hua; Tsai, Chang-Hai; Tsai, Fuu-Jen; Tsai, Henry J; Huang, Chih-Yang



Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins  

Microsoft Academic Search

Spinal muscular atrophy (SMA) is a neuro- degenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilit- ates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN

Gunter Meister; Dirk Bühler; Bernhard Laggerbauer; Monika Zobawa; Friedrich Lottspeich; Utz Fischer



GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein*  

PubMed Central

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin ?/? pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5?-3-O-(thio)triphosphate (GTP?S). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A. Barbara; Mitter, Richard; Svejstrup, Jesper Q.



GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein.  


We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin ?/? pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O-(thio)triphosphate (GTP?S). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase. PMID:21844196

Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara; Mitter, Richard; Svejstrup, Jesper Q



Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner  

Microsoft Academic Search

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress- activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in

Laurent Miccoli; Denis S. F. Biard; Isabelle Frouin; Francis Harper; Giovanni Maga; Jaime F. Angulo



Oxygen-Dependent Ubiquitination and Degradation of Hypoxia-Inducible Factor Requires Nuclear-Cytoplasmic Trafficking of the von Hippel-Lindau Tumor Suppressor Protein  

Microsoft Academic Search

It is becoming increasingly evident that the degradation of nuclear proteins requires nuclear-cytoplasmic trafficking of both the substrate proteins, as well as the E3 ubiquitin-ligases. Here, we show that nuclear- cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein (VHL) is required for oxygen- dependent ubiquitination and degradation of the alpha subunits of hypoxia-inducible factor (HIF-). VHL engages in a

Isabelle Groulx; Stephen Lee



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

PubMed Central

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

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



Analysis of the localization and topology of nurim, a polytopic protein tightly associated with the inner nuclear membrane.  


Nurim is an inner nuclear membrane (INM) protein that was first isolated in a visual screen for nuclear envelope-localizing proteins. Nurim lacks an N-terminal domain characteristic of other INM proteins examined to date and may represent a class of proteins that localize to the INM by a distinct mechanism. To further characterize this protein, we constructed nurim-green fluorescent protein fusions and analyzed aspects of localization, biochemistry, and membrane topology. Results from immunoprobing and protease protection assays together with other analyses indicate that nurim (total length of 262 residues) is a six transmembrane-spanning protein and contains a hairpin turn in its C-terminal transmembrane domain, resulting in the N and C termini residing on the same side of the membrane. A loop region between the fourth and fifth transmembrane domains is exposed toward the nucleoplasm and contains a region accessible for site-specific endoproteinase cleavage. In biochemical fractionation, nurim remained extremely tightly bound to nuclear fractions and was released in significant quantities only in the presence of 4 m urea. Under conditions in which nuclear lamins were completely extracted, a significant population of nurim remained resistant to solubilization. This tight binding requires the C-terminal region of the protein. DNase treatment only marginally influenced its retention characteristics in nuclei. Results from consideration of sequence alignments and identification of specific topological features of nurim indicate that it may possess enzymic function. These results are discussed with reference to the retention mechanism and possible nuclear function of nurim. PMID:15542857

Hofemeister, Helmut; O'Hare, Peter



Nuclear import of UBL-domain protein Mdy2 is required for heat-induced stress response in Saccharomyces cerevisiae.  


Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-?NES, but not Mdy2-?NLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress. PMID:23285234

Arhzaouy, Khalid; Ramezani-Rad, Massoud



The A-type lamins: nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.  


Mutations in the lamin A (LMNA) gene are associated with the tissue-specific diseases Emery-Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD-1B), dilated cardiomyopathy with conduction system disease (DCM-CD), and Dunnigan's familial partial lipodystrophy (FPLD). Lamins A and C, the products of the LMNA gene, are nuclear intermediate filament proteins and are the major structural components of the lamina network that underlies and supports the nuclear envelope. Nuclear fragility and mislocalization of the nuclear envelope protein emerin are two defects induced by a lack of the A-type lamins. These observations reveal that organization and structural integrity of the nucleus are critical factors in the origins of certain dystrophic and cardiovascular diseases. PMID:11709282

Mounkes, L C; Burke, B; Stewart, C L



Atypical I?B proteins - nuclear 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.



Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated ?-amylase genes  

Microsoft Academic Search

Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat a-amylase genes (a-Amyl\\/18 and a-Amy2\\/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300–430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised

Paul J. Rushton; Richard Hooley; Colin M. Lazarus



Monoclonal antibodies to a M r 68000 pore complex glycoprotein interfere with nuclear protein uptake in Xenopus oocytes  

Microsoft Academic Search

Using a monoclonal antibody (PI1) raised against mouse lymphocyte nuclear matrix fractions we have identified a N-acetylglucosamine (G1cNAc)-containing glycoprotein of Mr 68000 as a component of the nuclear pore complexes of Xenopus laevis oocytes. The antigenic determinant recognized by antibody PI1 comprises both the sugar moiety and protein sequences since, on the one hand, added G1cNAc competed effectively for antibody

Marie-Christine Dabauvalle; Ricardo Benavente; Nathalie Chaly



Towards a robust description of intrinsic protein disorder using nuclear magnetic resonance spectroscopy.  


In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein. PMID:21874206

Schneider, Robert; Huang, Jie-rong; Yao, Mingxi; Communie, Guillaume; Ozenne, Valéry; Mollica, Luca; Salmon, Loïc; Jensen, Malene Ringkjøbing; Blackledge, Martin



Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.  


Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment. PMID:23936203

Müller, Rebekka; Misund, Kristine; Holien, Toril; Bachke, Siri; Gilljam, Karin M; Våtsveen, Thea K; Rø, Torstein B; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit



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


AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic ? subunit and regulatory ? and ? 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 ?-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?2-binding protein. Artemis was found to co-immunoprecipitate with AMPK?2, and the co-localization of Artemis with AMPK?2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK?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?2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex. PMID:23044421

Nakagawa, Koji; Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie; Asaka, Masahiro; Takeda, Hiroshi; Kobayashi, Masanobu



Immunohistochemical localization of spermatid nuclear transition protein 2 in the testes of rats and mice.  


Transition protein 2 (TP2) of the rat was isolated by differential precipitation with trichloroacetic acid, chromatography over Bio-Rex 70, and preparative gel electrophoresis. A polyclonal rabbit antiserum was raised that did not cross-react with unrelated acid-soluble proteins from liver or testes. The antiserum was used to identify TP2-related proteins obtained from testes of mice, hamsters, guinea pigs, rabbits, and boars by Western blotting. Immunohistochemical techniques were used to localize TP2 in paraffin-embedded testis sections from mice and rats. In both species, TP2 was first detected in spermatids that had essentially completed the morphological change from a round to an elongate nucleus and that were undergoing chromosomal condensation (spermatids of step 13 in rat and step 12 in mouse). TP2 was retained in spermatid nuclei until early step 16 in the rat and step 14 in the mouse. Serial sections of rat testis exposed separately to antisera to TP1 and TP2 showed that the great majority of labeled tubules were reactive to both antisera. However, in occasional tubules, TP1 reactivity was retained in relatively late spermatids that were negative for TP2. Thus both TP1 and TP2 appear in the nucleus essentially simultaneously, in association with the beginning of chromatin condensation and at a point well after much of the nuclear shaping has occurred. PMID:8452928

Alfonso, P J; Kistler, W S



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


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



The geminivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity  

PubMed Central

Despite the large number of leucine-rich-repeat (LRR) receptor-like-kinases (RLKs) in plants and their conceptual relevance in signaling events, functional information is restricted to a few family members. Here we describe the characterization of new LRR-RLK family members as virulence targets of the geminivirus nuclear shuttle protein (NSP). NSP interacts specifically with three LRR-RLKs, NIK1, NIK2, and NIK3, through an 80-amino acid region that encompasses the kinase active site and A-loop. We demonstrate that these NSP-interacting kinases (NIKs) are membrane-localized proteins with biochemical properties of signaling receptors. They behave as authentic kinase proteins that undergo autophosphorylation and can also phosphorylate exogenous substrates. Autophosphorylation occurs via an intermolecular event and oligomerization precedes the activation of the kinase. Binding of NSP to NIK inhibits its kinase activity in vitro, suggesting that NIK is involved in antiviral defense response. In support of this, infectivity assays showed a positive correlation between infection rate and loss of NIK1 and NIK3 function. Our data are consistent with a model in which NSP acts as a virulence factor to suppress NIK-mediated antiviral responses.

Fontes, Elizabeth P.B.; Santos, Anesia A.; Luz, Dirce F.; Waclawovsky, Alessandro J.; Chory, Joanne



S100A6 mediates nuclear translocation of Sgt1: a heat shock-regulated protein.  


Sgt1 was originally identified in yeast as a suppressor of the Skp1 protein. Later, it was found that Sgt1 is present in plant and mammalian organisms and that it binds other ligands such as S100A6, a calcium-binding protein. In this work we show that in HEp-2 cells Sgt1 translocates to the nucleus due to heat shock. We also found that in HEp-2 cells with diminished level of S100A6, due to stable transfection with siRNA against S100A6, such translocation occurred at a much smaller scale in comparison with cells expressing a normal level of S100A6. Moreover, translocation of Sgt1 was observed in HEp-2 cells treated with thapsigargin instead of heat shock. In contrast thapsigargin was ineffective in cells with diminished level of S100A6. Thus, our results suggest that increase in intracellular concentration of Ca(2+), transduced by S100A6, is necessary for nuclear translocation of the Sgt1 protein. PMID:20213445

Prus, Wiktor; Filipek, Anna



Nuclear magnetic resonance of hyperpolarized fluorine for characterization of protein-ligand interactions.  


Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the (19)F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery. PMID:23020226

Lee, Youngbok; Zeng, Haifeng; Ruedisser, Simon; Gossert, Alvar D; Hilty, Christian



Nuclear Import and Dimerization of Tomato ASR1, a Water Stress-Inducible Protein Exclusive to Plants  

PubMed Central

The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an “NLS” of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.

Ricardi, Martiniano M.; Guaimas, Francisco F.; Gonzalez, Rodrigo M.; Burrieza, Hernan P.; Lopez-Fernandez, Maria P.; Estevez, Jose M.; Iusem, Norberto D.



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

NASA Astrophysics Data System (ADS)

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

Zerbetto, Mirco; Polimeno, Antonino; Meirovitch, Eva


Nuclear uptake control of NF-kappa B by MAD-3, an I kappa B protein present in the nucleus.  

PubMed Central

I kappa B proteins specifically inhibit the DNA binding of NF-kappa B/Rel transcription factors. An additional role as inhibitors of nuclear uptake was supposed by subcellular fractionation and enucleation experiments. Using indirect immunofluorescence labeling of cells, we show here that the DNA-binding p50 and p65 subunits of NF-kappa B, as well as the I kappa B protein MAD-3, all occur in the nucleus when overexpressed on their own. Nuclear uptake of p65 and, to a lesser extent of p50, was, however, suppressed when MAD-3 was coexpressed. Likewise, nuclear uptake of MAD-3 was blocked by overexpressed p65 or p50. This directly demonstrates that I kappa B is a nuclear uptake regulatory protein and that the various subunits of NF-kappa B can mutually control their access to the nucleus. In the presence of MAD-3, antibodies specific for peptides overlapping the nuclear location signal (NLS) sequences of p65 and p50 could not recognize their epitopes on NF-kappa B, suggesting that the I kappa B protein rendered the signals inaccessible for NLS receptors. Images

Zabel, U; Henkel, T; Silva, M S; Baeuerle, P A



Heterogeneous Nuclear Ribonucleoprotein A3 Is the Liver Nuclear Protein Binding to Age Related Increase Element RNA of the Factor IX Gene  

PubMed Central

Background In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes. Principal Finding We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser359, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser359. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable. Conclusions We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

Hamada, Toshiyuki; Kurachi, Sumiko; Kurachi, Kotoku



Nuclear Entry Mechanism of Rat PER2 (rPER2): Role of rPER2 in Nuclear Localization of CRY Protein  

PubMed Central

Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in both Drosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.

Miyazaki, Koyomi; Mesaki, Miho; Ishida, Norio



Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies*  

PubMed Central

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

Patsalo, Vadim; Yondola, Mark A.; Luan, Bowu; Shoshani, Ilana; Kisker, Caroline; Green, David F.; Raleigh, Daniel P.; Hearing, Patrick



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


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



Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity.  


Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response. PMID:9472026

Görner, W; Durchschlag, E; Martinez-Pastor, M T; Estruch, F; Ammerer, G; Hamilton, B; Ruis, H; Schüller, C



Minireview: Role of Protein Methylation and Demethylation in Nuclear Hormone Signaling  

PubMed Central

Nuclear hormone receptors (NRs) are transcription factors responsible for mediating the biological effects of hormones during development, metabolism, and homeostasis. Induction of NR target genes is accomplished through the assembly of hormone-bound NR complexes at target promoters and coincides with changes in histone modifications that promote transcription. Some coactivators and corepressors of NR can enhance or inhibit NR function by covalently modifying histones. One such modification is methylation, which plays important roles in transcriptional regulation. Histone methylation is catalyzed by histone methyltransferases and reversed by histone demethylases. Recent studies have uncovered the importance of these enzymes in the regulation of NR target genes. In addition to histones, these enzymes have nonhistone substrates and can methylate and demethylate NRs and coregulatory proteins in order to modulate their function. This review discusses recent progress in our understanding of the role of methylation and demethylation of histones, NRs, and their coregulators in NR-mediated transcription.

Wu, Susan C.; Zhang, Yi



Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1.  


Agrobacterium is a unique model system as well as a major biotechnological tool for genetic manipulation of plant cells. It is still unknown, however, whether host cellular factors exist that are limiting for infection, and whether their overexpression in plant cells can increase the efficiency of the infection. Here, we examined the effect of overexpression in tobacco plants of an Arabidopsis gene, VIP1, which encodes a recently discovered cellular protein required for Agrobacterium infection. Our results indicate that VIP1 is imported into the plant cell nucleus via the karyopherin alphadependent pathway and that elevated intracellular levels of VIP1 render the host plants significantly more susceptible to transient and stable genetic transformation by Agrobacterium, probably because of the increased nuclear import of the transferred-DNA. PMID:12124400

Tzfira, Tzvi; Vaidya, Manjusha; Citovsky, Vitaly



PML Is Critical for ND10 Formation and Recruits the PML-interacting Protein Daxx to this Nuclear Structure When Modified by SUMO1  

Microsoft Academic Search

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accu- mulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by iden- tifying proteins that are essential for this process using cells lines that lack individual ND10-associated pro- teins. We identified the adapter protein Daxx

Alexander M. Ishov; Alexey G. Sotnikov; Dmitri Negorev; Olga V. Vladimirova; Norma Neff; Tetsu Kamitani; Edward T. H. Yeh; Jerome F. Strauss III; Gerd G. Maul



Electron-nuclear interactions as probes of domain motion in proteins  

NASA Astrophysics Data System (ADS)

Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin-paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.

Shapira, Boaz; Prestegard, James H.



Electron-nuclear interactions as probes of domain motion in proteins  

PubMed Central

Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin–paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.

Shapira, Boaz; Prestegard, James H.



Dynamic correlation networks in human peroxisome proliferator-activated receptor-? nuclear receptor protein.  


Peroxisome proliferator-activated receptor-? nuclear receptor (PPAR-?) belongs to the superfamily of nuclear receptor proteins that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism. A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-?. Here we use normal-mode analysis of PPAR-? to characterize a network of dynamically coupled amino acids that link physiologically relevant binding surfaces such as the ligand-dependent activation domain AF-2 with the ligand binding site and the heterodimer interface. Multiple calculations were done in both the presence and absence of the agonist rosiglitazone, and the differences in dynamics were characterized. The global dynamics of the ligand binding domain were affected by the ligand, and in particular, changes to the network of dynamically correlated amino acids were observed with only small changes in conformation. These results suggest that changes in dynamic couplings can be functionally significant with respect to the transmission of allosteric signals. PMID:20496064

Fidelak, Jeremy; Ferrer, Silvia; Oberlin, Michael; Moras, Dino; Dejaegere, Annick; Stote, Roland H



The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors.  

PubMed Central

The HBx protein of hepatitis B virus (HBV) is a transcriptional activator that is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. Recently, we and others have shown that HBx stimulates the Ras-Raf-MAP kinase cascade, which leads to enhanced cell proliferation and the activation of transcription factors AP-1 and NF-kappa B. Other studies have shown that HBx can activate transcription by interacting directly with nuclear components of the transcription machinery. Therefore we examined the basis for the different reported activities of HBx. Here, we show that HBx is a complex protein, displaying independent activities in different intracellular locations. The intracellular distribution of HBx protein was first investigated using scanning confocal laser immunomicroscopy and by genetic studies. Our work has established that HBx expressed in cultured cells is found authentically in both the cytoplasm and the nucleus. HBx is not strongly associated with any intracellular structures, but some preferential accumulation was observed near the cell surface. Next, HBx variants were constructed containing a functional or mutant nuclear localization sequence. We show that when HBx is engineered to relocate exclusively to the nucleus, it no longer activates t