Biomedical subjects

Mitsuaki Yanagida

Publications and source records attributed to Mitsuaki Yanagida.

7 recordsLinked to original sources

PubMed2003

Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome.

Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.

Amino Acid Motifs↗

PubMed2003

Proteomics: advanced technology for the analysis of cellular function.

Proteomics developed initially from the decade-long study of comprehensive protein visualization on two-dimensional electrophoresis gels has been expanded by mass spectrometry and the growth in searchable sequence databases. Currently, by use of more sophisticated technology such as a combination of multidimensional chromatography and mass spectrometry, thousands of proteins can automatically be identified in a day along with semiquantitative information on differential-protein expression. As with differential gene expression by cDNA-chips, the differential-protein analysis is useful for monitoring and identifying proteins involved in various physiological changes in cells or organisms, although the analysis alone does not necessarily provide information regarding the cause of the change or the function of the proteins. However, proteomics also provides the tools to expand into more sophisticated biochemical approaches, such as the study of protein interactions that can be determined directly by performing a pull-down assay with a bait protein followed by mass spectrometric identification of the bound proteins. Proteomics, thus, is useful for both large-scale surveys of proteins and detailed studies of the functional relationships among the proteins of interest. Certainly this approach can be applicable to the assessment of amino acid adequacy and safety.

Animals↗

PubMed2002

A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics.

One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.

Amino Acid Sequence↗

PubMed2002

Functional proteomics; current achievements.

This review presents the current improvements in functional proteomic strategies and their research applications. Proteomics has emerged as an indispensable methodology for large-scale and high-throughput protein analyses in the post-genome era. Functional proteomics, the comprehensive analysis of proteins with special attention to their functions, is a powerful and useful approach for investigations in the life and medical sciences. Various methods have been developed for this purpose, expanding the field further. This important technology will not only provide a wealth of information on proteins, but also contribute synergistically to the understanding of life with other systematic technologies such as gene chips.

Protein Processing, Post-Translational↗

PubMed2002

Isolation and proteomic characterization of human Parvulin-associating preribosomal ribonucleoprotein complexes.

Human parvulin (hParvulin; Par14/EPVH) belongs to the third family of peptidylprolyl cis-trans isomerases that exhibit an enzymatic activity of interconverting the cis-trans conformation of the prolyl peptide bond, and shows sequence similarity to the regulator enzyme for cell cycle transitions, human Pin1. However, the cellular function of hParvulin is entirely unknown. Here, we demonstrate that hParvulin associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes, which contain preribosomal RNAs, at least 26 ribosomal proteins, and 26 trans-acting factors involved in rRNA processing and assembly at an early stage of ribosome biogenesis. Since an amino-terminal domain of hParvulin, which is proposed to be a putative DNA-binding domain, was alone sufficient to associate in principle with the pre-rRNP complexes, the association is probably through protein-RNA interaction. In addition, hParvulin co-precipitated at least 10 proteins not previously known to be involved in ribosome biogenesis. Coincidentally, most of these proteins are implicated in regulation of microtubule assembly or nucleolar reformation during the mitotic phase of the cell. Thus, these results, coupled with the preferential nuclear localization of hParvulin, suggest that hParvulin may be involved in ribosome biogenesis and/or nucleolar re-assembly of mammalian cells.

Animals↗

PubMed2002

Nuclear receptor function requires a TFTC-type histone acetyl transferase complex.

Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function.

Acetyltransferases↗

PubMedDate not supplied

Proteomic snapshot analyses of preribosomal ribonucleoprotein complexes formed at various stages of ribosome biogenesis in yeast and mammalian cells.

Proteomic technologies powered by advancements in mass spectrometry and bioinformatics and coupled with accumulated genome sequence data allow a comprehensive study of cell function through large-scale and systematic protein identifications of protein constituents of the cell and tissues, as well as of multi-protein complexes that carry out many cellular function in a higher-order network in the cell. One of the most extensively analyzed cellular functions by proteomics is the production of ribosome, the protein-synthesis machinery, in the nucle(ol)us--the main site of ribosome biogenesis. The use of tagged proteins as affinity bait, coupled with mass spectrometric identification, enabled us to isolate synthetic intermediates of ribosomes that might represent snapshots of nascent ribosomes at particular stages of ribosome biogenesis and to identify their constituents--some of which showed dynamic changes for association with the intermediates at various stages of ribosome biogenesis. In this review, in conjunction with the results from yeast cells, our proteomic approach to analyze ribosome biogenesis in mammalian cells is described.

Animals↗