Interaction of nucleolar phosphoprotein B23 with nucleic acids

Abstract

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

“…In all of these situations, at least one of two tryptophan (W) residues in positions 288 and 290 (WQWRKSL), which are necessary for the nucleolar localization of NPM1, shift into other amino acids 31. Of 23 patients who had mutant NPM1 in our study,14 (60.8%) patients had mutant allele A; 5 (21.7%), allele D; and 4 (17.4%), allele B. Our results showed a high frequency of allele D of the mutant NPM1 gene among Iranian AML patients.…”

“…Several functions have already been described for NPM1 protein, including binding to nucleic acids,14 regulation of centrosome duplication15 and also ribosomal function 16. Furthermore, NPM1 binds to several proteins, like p53 and proteins that react to and regulate p53 (like Rb,17 p19ARF18 and HDM219).…”

Down-regulation of nucleophosmin/B23 during retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

“…Nucleophosmin/B23 has been ascribed a number of diverse properties, including a potential role in proliferation due to its rapid increase in synthesis during mitogenic stimulation (Feuerstein and Mond 1987b); a role as a cytoplasmic/nuclear shuttle protein (Borer et al, 1989); has DNA binding activity (Dumbar et al, 1989); relieves transcription repression by YY1 (Inouye and Seto, 1994); binds the HIV Type 1 Rev protein (Fankhauser et al, 1991), the human Tcell leukemia virus-1-Rex protein (Adachi et al, 1993), and another cell cycle-regulated nucleolar oncoprotein p120 (Valdez et al, 1994); is a substrate of protein kinase C (Beckmann et al, 1992), mitotic cdc2 kinase (Peter et al, 1990), and casein kinase II (Pfa and Anderer, 1988); can be ADP-ribosylated (Leitinger and Wesierska-Gadek, 1993). Nucleophosmin/B23 is of potential interest because it is involved in at least three distinct forms of hematologic malignancy.…”

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

“…37 Nucleic acid binding was later tested in vitro on both single and double stranded targets. It emerged that NPM1 binds nucleic acids independently from their sequence and with a preference for single-stranded DNA and RNA, over double-stranded DNA.…”