No abstract

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N-and Cterminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N-and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acidbinding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.Ribosome assembly is a multistep process that utilizes numerous proteins and small nucleolar RNAs (1, 2). One candidate for a ribosome assembly factor is an abundant protein called B23 (also known as nucleophosmin/NPM (3), NO38 (4), or numatrin (5)) whose location, abundance, and multiple activities suggest that it plays a major role in ribosome biogenesis. This is supported by the ability of protein B23 to bind nucleic acids (6, 7) and by its association with maturing preribosomal ribonucleoprotein particles (4,8,9). Treatment of cells with drugs that inhibit preribosomal RNA processing or synthesis (10, 11) causes translocation of B23 to the nucleoplasm, which further suggests its presence in nascent preribosomal particles. Finally, protein B23 possesses intrinsic ribonuclease activity that has been implicated in the processing of preribosomal RNA in the internal transcribed spacer region 2 region (12, 13).Protein B23 interacts with other nucleolar proteins, including nucleolin (14), protein p120 (15), and the HIV-1 Rev protein (16). Its ability to shuttle between the nucleus and cytoplasm (17), bind nuclear localization signal containing peptides (18), and stimulate import of proteins into the nucleus (18) suggested a role in nuclear import. The latter activity might be explained by its ability to act as a molecular chaperone (19). In normal cells, this activity may aid in the transport of ribosomal or other nucleolar proteins from their site of synthesis into the nucleus or nucleolus. Alternatively, protein B23 could serve as a chaperone in preventing aggregation of proteins in the very crowded environment of the nucleolus during ribosome assemb...

As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.

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.

Protein B23/nucleophosmin is a multifunctional protein that plays roles in ribosome biogenesis, control of centrosome duplication, and regulation of p53 expression. A yeast two-hybrid screen was performed in a search for interaction partners of B23. The complementary DNA for a highly acidic protein, nucleoplasmin 3 (NPM3), was found in multiple positive clones. Protein NPM3 and its interaction with B23 were further characterized. Endogenous B23 was able to be co-immunoprecipitated with NPM3, and this complex was resistant to ribonuclease treatment and high concentrations of salt. The N-terminal 35-90 amino acids of B23 were found to be required for their interaction. Separate co-immunoprecipitation studies of B23 and NPM3 suggested the existence of two different complexes, one containing B23 and 28 S ribosomal RNA (rRNA) and another composed of B23, NPM3, and other proteins, but no RNA. NPM3 was localized in the nucleolus, and its nucleolar localization depended on active rRNA transcription. In the cells overexpressing NPM3, there were decreased rates of pre-rRNA synthesis and processing. Overexpression of a mutant of NPM3 that did not interact with B23 did not alter pre-rRNA synthesis and processing, suggesting that the interaction of NPM3 with B23 plays a role in the ribosome biogenesis.Ribosome biogenesis in eukaryotic cells is a multistep process that takes place primarily in the nucleolus where the individual stages of assembly correlate with specific subclasses of ultrastructures (1-4). The process begins with transcription of the ribosomal DNA at the border between the fibrillar center and the dense fibrillar components of the nucleolus. The product of transcription, 47 S pre-ribosomal RNA (pre-rRNA) 1 in mammals, is processed into smaller pre-rRNA intermediates, which finally become 28, 5.8, and 18 S rRNA. The nascent pre-ribosomal particles of the dense fibrillar components eventually mature into granular components, with ribosomal proteins added at various steps in the process. Numerous nonribosomal proteins and small nucleolar RNA participate in these steps.Protein B23 (NPM1, nucleophosmin) is an abundant nucleolar non-ribosomal protein whose locations and multiple activities suggest it plays a role in ribosome biogenesis. This protein is primarily localized to the granular component region with lesser amounts in the dense fibrillar components of the nucleolus (5-8). The nucleolar localization of B23 is dependent on the presence of active rDNA transcription (9), and it is found in association with maturing pre-ribosomal RNP particles (10, 11). In vitro experiments indicate B23 has nucleic acid binding activity and ribonuclease activities (12-17). The nucleic acid binding activity has been mapped to its C-terminal end (17,18) and is believed to be important in its nucleolar localization (15).In support of this, a splicing variant, B23.2, in which the C-terminal 35-amino acid sequence is absent, exists both in the nucleoplasm and cytoplasm (15,17). More recent studies provide evidence for a direct role ...

Nucleolar protein B23 is a putative ribosome assembly factor with a high affinity for peptides containing nuclear localization signals (NLSs). The interactions of various NLS-containing peptides with two B23 isoforms (B23.1 and B23.2) were examined using equilibrium dialysis and Scatchard analyses. The KD for protein B23 binding to a peptide containing the SV40 T-antigen NLS sequence was approximately 1 microM with a stoichiometry of 1:1 (peptide:protein). No significant differences were seen between the two B23 isoforms in their affinities for any of the peptides tested. Binding by a reverse sequence SV40 T-NLS peptide showed a nonlinear Scatchard plot: this peptide was unable displace the correct sequence peptide, suggesting that the reverse sequence peptide binds to a different site on the protein. A peptide containing the sequence required for nucleolar localization of the HIV-1 Rev protein had an affinity for B23 approximately 10-fold greater than that of the SV40 T-NLS. However, with a sequence sufficient only for Rev location in the nucleoplasm, the affinity for B23 was diminished to a level between that of the longer Rev sequence and the SV40 T-NLS. In competition binding assays, the Rev NLS peptide was able to displace the SV40 T NLS, indicating that both peptides bind to the same site on protein B23. There was no detectable binding to protein B23 by a peptide containing the bipartite NLS of nucleoplasmin. Phosphorylation of protein B23 by casein kinase II enhanced its affinity for the SV40 T- and Rev-derived peptides approximately 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

IntroductionMajor depressive disorder (MDD) has a lifetime prevalence of 16% in the United States.1 Antidepressant drugs are the most used intervention for those with a diagnosis of depressive disorders, but the road to remission is long and uncertain, with 40% of patients never reaching full remission and at least 15% not experiencing any symptomatic improvements.2 Elucidating the biological bases of MDD is likely to provide novel targets for the development of more effective drugs, or at the very least, adjunctive treatments for existing antidepressants that increase the chance of remission.Speculation that norepinephrine plays a role in depressive disorders dates back to the early 1950s, and research since then increasingly supports this. The locus coeruleus (LC) in the pontine brainstem contains the cell bodies of the major source of norepinephrine in the brain and has been the subject of numerous investigations regarding the neuropathology of MDD.3 The human LC is an area with very high densities of radioligand binding of antidepressant drugs to monoamine oxidase, 4 the norepinephrine transporter 5 and the serotonin transporter.6 Numerous postmortem studies demonstrate abnormal neurochemistry of the noradrenergic LC in people with MDD and in people who died by suicide. A role of Background: Norepinephrine and glutamate are among several neurotransmitters implicated in the neuropathology of major depressive disorder (MDD). Glia deficits have also been demonstrated in people with MDD, and glia are critical modulators of central glutamatergic transmission. We studied glia in men with MDD in the region of the brain (locus coeruleus; LC) where noradrenergic neuronal cell bodies reside and receive glutamatergic input. Methods: The expression of 3 glutamate-related genes (SLC1A3, SLC1A2, GLUL) concentrated in glia and a glia gene (GFAP) were measured in postmortem tissues from men with MDD and from paired psychiatrically healthy controls. Initial gene expression analysis of RNA isolated from homogenized tissue (n = 9-10 pairs) containing the LC were followed by detailed analysis of gene expressions in astrocytes and oligodendrocytes (n = 6-7 pairs) laser captured from the LC region. We assessed protein changes in GFAP using immunohistochemistry and immunoblotting (n = 7-14 pairs). Results: Astrocytes, but not oligodendrocytes, demonstrated robust reductions in the expression of SLC1A3 and SLC1A2, whereas GLUL expression was unchanged. GFAP expression was lower in astrocytes, and we confirmed reduced GFAP protein in the LC using immunostaining methods. Limitations: Reduced expression of protein products of SLC1A3 and SLC1A2 could not be confirmed because of insufficient amounts of LC tissue for these assays. Whether gene expression abnormalities were associated with only MDD and not with suicide could not be confirmed because most of the decedents who had MDD died by suicide. Conclusion: Major depressive disorder is associated with unhealthy astrocytes in the noradrenergic LC, characterized here by a reduction in a...

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.