Phase Separation of Intrinsically Disordered Nucleolar Proteins Relate to Localization and Function.

The process of phase separation allows for the establishment and formation of subcompartmentalized structures, thus enabling cells to perform simultaneous processes with precise organization and low energy requirements. Chemical modifications of proteins, RNA, and lipids alter the molecular environment facilitating enzymatic reactions at higher concentrations in particular regions of the cell. In this review, we discuss the nucleolus as an example of the establishment, dynamics, and maintenance of a membraneless organelle with a high level of organization.

Profiling Cell Lines Nuclear Sub-proteome.

Proteins are very dynamic within the cell and their localization and trafficking between subcellular compartments are critical for their correct function. Indeed, the abnormal localization of a protein might lead to the pathogenesis of several diseases. The association of cell fractionation methods and mass spectrometry based proteomic methods allow both the localization and quantification of proteins in different sub-compartments. Here we present a detailed protocol for enrichment, identification, and quantitation of the nuclear proteome in cell lines combining nuclear subproteome enrichment by differential centrifugation and high-throughput proteomics.

Proteomic characterization of the human FTSJ3 preribosomal complexes.

In eukaryotes, ribosome biogenesis involves excision of transcribed spacer sequences from the preribosomal RNA, base and ribose covalent modification at specific sites, assembly of ribosomal proteins, and transport of subunits from the nucleolus to the cytoplasm where mature ribosomes engage in mRNA translation. The biochemical reactions throughout ribosome synthesis are mediated by factors that associate transiently to the preribosomal complexes. In this work, we describe the complexes containing the human protein FTSJ3. This protein functions in association with NIP7 in ribosome synthesis and contains a putative RNA-methyl-transferase domain (FtsJ) in the N-terminal region and two uncharacterized domains in the central (DUF3381) and C-terminal (Spb1_C) regions. FLAG-tagged FTSJ3 coimmunoprecipitates both RPS and RPL proteins, ribosome synthesis factors, and proteins whose function in ribosome synthesis has not been demonstrated yet. A similar set of proteins coimmunoprecipitates with the Spb1_C domain, suggesting that FTSJ3 interaction with the preribosome complexes is mediated by the Spb1_C domain. Approximately 50% of the components of FTSJ3 complexes are shared by complexes described for RPS19, Par14, nucleolin, and NOP56. A significant number of factors are also found in complexes described for nucleophosmin, SBDS, ISG20L2, and NIP7. These findings provide information on the dynamics of preribosome complexes in human cells.

The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin.

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

An improved method to obtain a soluble nuclear fraction from embryonic brain tissue.

This paper describes modifications of the standard methods for obtaining a soluble nuclear fraction from embryonic brain tissue. The main improvements are: (1) the inclusion of a low speed centrifugation step to prevent the appearance of high density contaminants, (2) a sucrose density gradient to remove perinuclear mitochondria and ER membranes and (3) a protein extraction approach which significantly enhances protein yield. To demonstrate the effectiveness of the method, pellets were analyzed by light and electron microscopy and purity of the soluble extracts was immunologically tested. Finally, to illustrate the applicability of this approach, the induction of the transcription factor HIF-1 (hypoxia-inducible factor-1) was assessed by Western blot using soluble nuclear fractions and by immuno-electron microscopy using purified nuclear fractions, both obtained from the optic lobes of chick embryos. In conclusion, the procedure presently described appears to be reliable and convenient for obtaining a pure soluble nuclear fraction from a discrete amount of embryonic brain tissue.

A partially purified putative iron P type-ATPase mediates Fe3+-transport into proteoliposome.

We report that two fractions containing proteins from rat hepatocyte nuclei, obtained by nondenaturing gel electrophoresis, were able to bind iron and ATP, and to hydrolyze ATP. Electroelution of these two active fractions followed by SDS-PAGE analysis showed an identical protein pattern, each one containing four proteins in a range of 62-80 kDa. Phosphorylated protein bands were also detected in acid gel and disappeared after treatment with hydroxylamine/acetate or KOH, and upon chasing with cold ATP. A proteoliposome system, made by the incorporation of these partially purified protein fractions into phosphatidylcholine vesicles, carried out Fe(3+)-citrate uptake in a Mg(2+)-ATP-dependent way; Fe(3+) accumulation increased with time reaching a plateau in 30 min. Iron uptake was not supported by AMP-PNP, was partially inhibited by orthovanadate and was not affected by a mix of specific inhibitors of known ATPases. These results support our previous hypothesis that a putative nuclear membrane Fe(3+)-ATPase is involved in nuclear iron homeostasis.

Expression of myelin basic protein in two oligodendroglial cell lines is modulated by apotransferrin through different transcription factors.

We have shown that apotransferrin (aTf) promotes the differentiation of two oligodendroglial cell (OLGc) lines, N19 and N20.1, representing different stages of OLGc maturation. Although in both cell lines aTf promoted myelin basic protein (MBP) expression, an increase in cAMP levels and CREB phosphorylation was observed only in the less mature cells (N19), suggesting that the maturation induced by aTf is achieved probably through different signaling pathways. We transfected both cell lines with the proximal region of the human MBP promoter fused to the lacZ reporter gene. In both transfected cell lines, addition of aTf produced an activation of the promoter. To elucidate the mechanisms involved in this action, Western blot analysis, EMSAs, and RT-PCR were performed for different transcription factors involved in mbp regulation. In the N20.1 line, treatment with aTf increased the expression and the DNA-binding capacity of thyroid hormone (TH) receptors, Sp1, and nuclear factor-kappaB (NFkappaB). For these cells we found that an inductor of NFkappaB (tumor necrosis factor-alpha) promoted MBP messenger synthesis, whereas mithramycin, a specific inibitor of Sp1, and a cAMP analog (db-cAMP) inhibited its transcription. In the N19 cell line, aTf stimulated NF-I and NFkappaB activation, but, aside from aTf, only db-cAMP induced mbp transcription. These data suggest that, depending on the OLGc maturational stage, aTf modulates MBP expression and OLGc differentiation through different signaling pathways and different transcription factors.

Entamoeba histolytica: functional characterization of the -234 to -196 bp promoter region of the multidrug resistance EhPgp1 gene.

The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.

Identification of nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins.

An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.

Echinococcus granulosus: preparation of protein extracts from protoscolex nuclei for mobility-shift assays.

Nuclei from Echinococcus granulosus protoscolices were isolated from infected sheep. Protein extracts were prepared for analysis of DNA-protein interactions involving specific transcriptional regulatory factors. Gel mobility-shift assays were done using a heterologous probe containing binding sites for widespread transcription factors. A fragment of the promoter of GATA-1 transcription factor from the chicken was selected. When nuclear extracts from E. granulosus protoscolices were assayed a specific band shift was observed. The methodologies developed in this study could provide an important contribution for the characterization of the DNA-protein interactions involved in transcriptional regulation within the context of recent developments in the molecular biology of this parasite.

Differential genomic susceptibility in malignancy correlates with changes in ATATAT DNA-binding proteins.

Accesibility to DNA in the nucleus is important for the regulation of gene expression and for the effect of DNA-modifying drugs. We have now studied differential genome susceptibility in normal melanocytes and the corresponding malignant melanoma. DNA hypersensitivity assays revealed a markedly lesser degradation in melanoma nuclei compared to that in melanocytes. Cross-linking of DNA to nuclear proteins by ultraviolet light showed a cell-type dependent inverse correlation of genomic susceptibility with binding of (dA.dT) (dA.dT) sequences, compared to that shown with (dG.dC) (dG.dC), regardless of methylation in cytosines. Exposure to cholera toxin partly reversed genomic susceptibility and increased DNA/protein cross-linking in melanocytes. In contrast, melanoma cells showed decreased DNA/protein interactions and greater genome susceptibility after exposure to cholera toxin or okadaic acid. Our data suggest that a molecular mechanism for differential genome exposure in cancer cells involves a modified expression of sequence-specific DNA-binding proteins.

Analysis of supra-nucleosome particles from unfertilized eggs of sea urchins.

Two discrete supranucleosomal particles that differ in their electrophoretic migration on 1% agarose gels were isolated from unfertilized sea urchin eggs. Both particles contain the same complement of cleavage stage (CS) chromosomal proteins, which is identical to the complete set of basic proteins isolated directly from chromatin by extraction with 0.25 N HCl. DNA fragments between 210 and 1500 bp were found in both particles, and the basic unit of DNA repeat length determined by micrococcal nuclease digestion was 126 +/- 3 bp. The isolated nucleoparticles are electrophoretically stable over a wide range of DNA sizes (126-1500 bp) indicating that their structure is maintained by internal interactions among the CS chromosomal proteins, previously designated CS A through CS G. Based on these results we conclude that the CS chromosomal proteins are functionally equivalent to classical histones in their ability to direct higher ordered structures of chromatin.