Interaction between nucleophosmin and HBV core protein increases HBV capsid assembly

Host factors are involved in Hepatitis B virus (HBV) genome replication and capsid formation during the viral life cycle. A host factor, nucleophosmin (B23), was found to bind to HBV core protein dimers, but its functional role has not been studied. This interaction promoted HBV capsid assembly and decreased the degree of capsid dissociation when subjected to denaturant treatments in vitro. In addition, inhibition of B23 reduced intracellular capsid formation resulting in a decrease of HBV production in HepG2.2.15 cells. These results provide important evidence that B23 acts on core capsid assembly via its interaction with HBV core dimers.     

Overexpression of the nucleolar protein nucleophosmin/B23 in collagen lattice-cultured fibroblasts: Potential role in the control of protein synthesis

Fibroblasts cultivated in tridimensional collagen lattices exhibit a downregulation of protein synthesis, related to decreased ribosomal RNA (rRNA) content and half life, when compared to monolayer cultivated cells. The involvement in this process of nucleophosmin/B23, a nucleolar phosphoprotein with ribonuclease properties, was checked. We compared production of nucleophosmin/B23 in monolayer and collagen lattice cultured fibroblasts. A significant increase of nucleophosmin/B23 mRNA levels was noticed in lattice-cultured fibroblasts vs monolayers (+154%, p < 0.05). A concomitant enhancement of nucleolar nucleophosmin/B23 content was found (+112%, p < 0.001). Simultaneously, ribonuclease activity contained in nucleolar extracts from collagen lattice-cultured fibroblasts was significantly increased (+54%, p < 0.01). These data demonstrate that extracellular collagen matrix induces the overexpression of nucleophosmin/B23, and suggest that the regulation of protein syntheses in collagen lattice cultures may be explained, at least partly, by an increased degradation of neosynthesized rRNAs dependent on nucleophosmin.     

New centromeric component CENP-W is an RNA-associated nuclear matrix protein that interacts with nucleophosmin/B23 protein

CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.     

Mutation of cysteine 21 inhibits nucleophosmin/B23 oligomerization and chaperone activity

Nucleophosmin (NPM/B23) is a multifunctional nucleolar protein to which both tumor-suppressor and oncogenic functions have been attributed. NPM/B23 has a variety of binding partners including ribosomes, nucleic acids, the centrosome and tumor suppressors such as p53 and p19ARF. These disparate functions are likely due to its ability to oligomerize and display molecular chaperone activity. In this report we identify a single amino acid residue, Cys²¹, of nucleophosmin as important for the oligomerization and chaperone activity. Mutation of Cys²¹ to aromatic hydrophobic residues (e.g., Phe or Try), but not to a conserved polar residue (e.g., Ser) inhibited the pentameric oligomerization of NPM/B23. However, only Phe substitution of Cys²¹ drastically inhibited NPM/B23 chaperone activity. Interestingly, expression of Cys21Phe mutant in MCF7 cells demonstrated that this mutant protein does not co-polymerize with endogenous wild-type NPM/B23 and acts as negative dominant by destabilizing the endogenous dimer, trimer oligomerization. Taken together, the results in this study identify Cys²¹ as critical residue for NPM/B23 oligomerization and chaperone functions. In addition, Cys²¹ mutant provide a strong link between the oligomerization and chaperone functions of NPM/B23.     

Quantitative analysis of the interaction between the envelope protein domains and the core protein of human hepatitis B virus

Interaction between preformed nucleocapsids and viral envelope proteins is critical for the assembly of virus particles in infected cells. The pre-S1 and pre-S2 and cytosolic regions of the human hepatitis B virus envelope protein had been implicated in the interaction with the core protein of nucleocapsids. The binding affinities of specific subdomains of the envelope protein to the core protein were quantitatively measured by both ELISA and BIAcore assay. While a marginal binding was detected with the pre-S1 or pre-S2, the core protein showed high affinities to pre-S with apparent dissociation constants (K(D)(app)) of 7.3+/-0.9 and 8.2+/-0.4microM by ELISA and BIAcore assay, respectively. The circular dichroism analysis suggested that conformational change occurs in pre-S through interaction with core protein. These results substantiate the importance of specific envelope domains in virion assembly, and demonstrate that the interaction between viral proteins can be quantitatively measured in vitro.     

Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.     

Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23

The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19(Arf), we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19(Arf) associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19(Arf); however, a significant proportion of p19(Arf) associates with NPM. The interaction between p19(Arf) and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19(Arf) and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19(Arf) associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19(Arf) from entering into 2- to 5-MDa particles, and overrides the ability of p19(Arf) to retard rRNA processing.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.     

Involvement of nucleophosmin/B23 in the cellular response to curcumin

Nucleophosmin (NPM/B23) is a nucleolar phosphoprotein involved in cellular response to many different stimuli. Herein, we studied the molecular mechanism of NPM/B23 induction by curcumin, a natural AP-1 inhibitor with antitumor properties. Exposure to 5-30 μM curcumin significantly and dose-dependently increased the level of NPM/B23 in non-transformed NIH 3T3 cells but not HeLa cells and F9 cells. Besides, the transformed F9 and HeLa cells are more sensitive to curcumin-induced cell death and growth inhibition than NIH 3T3 cells. Overexpression of c-Jun, but not c-Fos, decreased ∼40% of NPM/B23 and enhanced the sensitivity of NIH 3T3 cells to 30 μM curcumin. Furthermore, down-regulation of NPM/B23 by transfection with NPM/B23 antisense plasmid enhanced the sensitivity to curcumin-induced cell death and growth inhibition. These results indicated that NPM/B23 expression regulates cellular sensitivity to curcumin. Besides, NPM/B23 knockdown may facilitate as a novel strategy to promote the sensitivity of cancer cells to curcumin.     

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.     

Physical and functional interactions between hnRNP K and PRMT family proteins

The mechanism underlying the protein-protein interaction of hnRNP K and PRMT family proteins is unclear. We examined and confirmed the arginine methylation of hnRNP K protein by PRMT1, not CARM1, via their direct binding. We also studied hnRNP K protein complexes containing CARM1, as well as PRMT1, using co-immunoprecipitation analysis. PRMT family proteins might be involved in the regulation of hnRNP K functions in nuclear receptor coactivator, transactivation, and p21 gene and protein expressions. We believe these observations will help provide insights into the regulation of hnRNP K protein functions via the recruitment of its associated proteins, including its arginine methylation-modifying proteins.

Structured summary

MINT-6803853: hnRPK, (uniprotkb:P61978) binds (MI:0407) to PRMT1 (uniprotkb:Q99873) by pull down (MI:0096)MINT-6803884: hnRPK, (uniprotkb:P61978) physically interacts (MI:0218) with CARM1 (uniprotkb:Q86X55) by anti tag coimmunoprecipitation (MI:0007)MINT-6803869: hnRPK, (uniprotkb:P61978) physically interacts (MI:0218) with PRMT1 (uniprotkb:Q99873) by anti tag coimmunoprecipitation (MI:0007)MINT-6803939: hnRPK, (uniprotkb:P61978) binds (MI:0407) to PRMT2 (uniprotkb:P55345) by pull down (MI:0096)MINT-6803929: hnRPK, (uniprotkb:P61978) binds (MI:0407) to RMT (uniprotkb:P38074) by pull down (MI:0096)MINT-6803896: hnRPK, (uniprotkb:P61978) binds (MI:0407) to PRMT3 (uniprotkb:O60678) by pull down (MI:0096)MINT-6803834: PRMT1 (uniprotkb:Q99873) methylates (MI:0213) hnRPK, (uniprotkb:P61978) by methyltransferase assay (MI:0515)     

Nucleolar protein B23 has molecular chaperone activities

Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.     

The nucleocapsid protein of SARS-associated coronavirus inhibits B23 phosphorylation

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is responsible for SARS infection. Nucleocapsid (N) protein of SARS-CoV encapsidates the viral RNA and plays an important role in virus particle assembly and release. In this study, the N protein of SARS-CoV was found to associate with B23, a phosphoprotein in nucleolus, in vitro and in vivo. Mapping studies localized the critical N sequences for this interaction to amino acid residues 175-210, which included a serine/arginine (SR)-rich domain. In vitro phosphorylation assay showed that the N protein inhibited the B23 phosphorylation at Thr199.     

Analysis of the proliferative activity of a cell using new monoclonal antibodies to nucleolar protein B23/nucleophosmin

Nowadays, antinucleolar antibodies are widely used for exploration of the nucleolar organization and molecular mechanisms of ribosome production. Here we have described a new monoclonal antibody against the major nucleolar phosphoprotein B23/nucleophosmin (3C9) that is involved in the terminal stages of ribosome production. It is used to examine immunocytochemical peculiarities of the nucleolus in terms of the cell proliferative status and also during mitosis. In human peripheral blood lymphocytes, activated for proliferation with phytohaemagglutinin (PHA), PHA stimulation of lymphocytes was shown to result in accumulation of protein B23 in augmentative nucleoli. A comparative study of 3C9 and two other anti-B23 antibodies 20B2 and anti-B23 by Western blots and indirect immunofluorescence favored the idea that 3C9 cross-reacted with the major isoform of B23, B23.1, that have an apparent molecular weight of 40 kDa.