Peptidyl-prolyl cis-trans isomerase (PPIase) catalyses the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and has been shown to accelerate the refolding of several proteins in vitro. Its activity has been detected in yeast, insects and Escherichia coli as well as in mammals, and it is though to be essential for protein folding during protein synthesis in the cell. We purified PPIase from pig kidney and found that its amino-acid sequence is identical to that reported for bovine cyclophilin, a protein known to bind the immunosuppressive drug, cyclosporin A (ref. 5). To investigate the functional relationship between PPIase and cyclophilin we examined the effect of cyclosporin A on PPIase activity and found that it was inhibitory. Thus we propose that the peptidyl-prolyl cis-trans isomerizing activity of PPIase may be involved in events, such as those occurring early in T-cell activation, that are suppressed by cyclosporin A.

Peptidylprolyl-cis-trans-isomerase (PPIase) is thought to be essential for protein folding in the cell. Two forms, a and b, of PPIase and their corresponding genes were isolated from Escherichia coli cells. Despite their insensitivity to cyclosporin A (CsA), both amino acid sequences were homologous and related to that of pig cyclophilin, a protein that has PPIase activity sensitive to CsA (Takahashi et al., 1989). PPIase a is found to be identical with the E. coli ORF 190 gene product that was sequenced by Kawamukai et al. (1989) and overexpressed by Liu and Walsh (1990). It is translocated into E. coli periplasmic space with the signal sequence. PPIase b lacks a hydrophobic amino acid stretch which could serve as a signal sequence or a transmembrane domain, and it is detected mainly in the bacterial cytoplasm. These findings indicate that proteins with the ability to assist folding of various polypeptides are located on both sides of the inner membrane. Thus, we propose that the folding of some exported proteins may be catalyzed by the periplasmic proline isomerase and, in turn, that some proteins which have isomerized may not be translocated efficiently.

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.The nucleolus serves many functions (4, 23, 36, 51); however, its most prominent function remains ribosome biogenesis. This process comprises rRNA gene transcription; processing of the 47S pre-rRNA to mature 18S, 5.8S, and 28S rRNAs; and assembly of preribosomal particles (21). Ribosome biogenesis is spatially organized in distinct compartments within the nucleolus. Transcriptionally active ribosomal genes are thought to be situated at either the boundaries of the fibrillar centers (FCs) and dense fibrillar components (DFCs) or in DFCs (45), whereas both the pre-rRNA processing and the assembly of preribosomal particles occur in the DFCs and granular components (GCs) (46). Ribosome biogenesis is regulated at multiple levels, including the transcription of ribosomal genes and the phosphorylation, methylation, and acetylation of component nucleolar factors, plus the trafficking and interaction of these factors (30).Proteasomal regulation has been implicated in many processes, including cell cycle progression, transcription, and antigen processing (24) (18,34,58). Indirect evidence has hinted at a possible role for the ubiquitin-proteasome system (UPS) in ribosomal biogenesis. Two different ubiquitination patterns have been reported for the late processing factor B23 (25,48). Several other ribosomal factors may be ubiquitinated, as suggested by a recent proteomic analysis in yeast (37). It has also been known for many years that two ubiquitin precursors are ribosomal fusion proteins (5,16,47). A large ribosomal subunit protein (L28) forms the most abundant ubiquitin-protein conjugate in yeast, and this modification is essential for ribosome function and efficient translation (53). A possible role for ubiquitin in nucleolus disassembly was also suggested (54). In addition, a ubiquitin ligase is known to regulate the processing and nuclear export of rRNA, as well as tRNA and mRNA in yeast (35). Finally, a temperature-sensitive point mutation of the Cic1p/Nsa3p yeast protein, an adaptor for the 26S proteasome, associates with early pre-60S particles (49), and inhibits both the synthesis of the mature 5.8S and 25S rRNAs and the releas...

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.

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.

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I. Introduction 288 II. Transcription and Processing of the Pre‐rRNA in the Ribosome Biogenesis of Yeast Cells: A Model for Eukaryotic Cells 290 III. Trans‐Acting Factors Involved in the Pre‐rRNA Processing and Assembly in Yeast Cells 291 A.  Trans‐Acting Proteins That Act with snoRNAs 291 B.  Trans‐Acting Factors Involved in the rRNA Cleavages 293 C.  Trans‐Acting Factors Involved in the rRNA Editing and Conformational Rearrangement 296 IV. Isolation and Proteomic Characterization of Pre‐rRNP Complexes Formed During Ribosome Biogenesis in Yeast Cells 296 A.  Experimental Approaches to Characterize Pre‐rRNP Complexes 296 B.  90S Pre‐rRNP Complexes Formed at Very Early Stages of Ribosome Biogenesis 299 C.  Pre‐rRNP Complexes Formed at Early/Middle Stages of Ribosome Biogenesis 302 D.  Pre‐rRNP Complexes Formed at Later Stages of Ribosome Biogenesis 303  V. Isolation and Proteomic Characterization of Pre‐rRNP Complexes Involved in Mammalian Ribosome Biogenesis 304 A.  Ribosome Biogenesis in Mammalian Cells 304 B.  Proteomic Analysis of the Pre‐rRNP Complexes Associated with Nucleolin: A Major Nucleolar Protein 305 C.  Reverse‐Tagging Methodology Applied to Human Trans‐Acting Proteins Involved in Ribosome Biogenesis 310 D.  Isolation and Proteomic Analysis of Human Parvulin‐Associating Pre‐rRNP Complexes 310  VI. Proteomic Analysis of the Nucleolus of Mammalian Cells 312 VII. Conclusions 312 VIII. Abbreviations 314 References 314 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. © 2003 Wiley Periodicals, Inc., Mass Spec Rev 22:287–317, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10057

IntroductionHsp90 is an abundant, evolutionarily conserved molecular chaperone whose function depends on its ability to bind and hydrolyze ATP. Through an ATPase cycle, Hsp90 facilitates proper folding of "client" proteins, thereby regulating their stability, protein interactions, intracellular trafficking, and functions. 1,2 To fulfill these functions, Hsp90 interacts with its cofactors and cochaperones including Hsp70, immunophilins, and p23, to form the Hsp90-based chaperone complex. 1,2 Natural compounds such as geldanamycin and radicicol bind the ATP-binding pocket of Hsp90 and disrupt its chaperone function. 3,4 Hsp90 is required for function and stability of diverse signal transduction proteins including oncogenic proteins such as ErbB2 and Raf-1. [2][3][4][5][6][7] Hence, the chaperone is an attractive target for cancer therapeutics. Indeed, Hsp90 inhibitors show antitumor activities in preclinical models, and geldanamycin analogues such as 17-allylamino-17-demethoxygeldanamycin (17-AAG) are currently undergoing clinical trials. 3,4 Importantly, Hsp90 inhibitors sensitize tumor cells to various genotoxic agents used for standard cancer therapeutics, including DNA crosslinkers, 8,9 ionizing radiation, 10 and replication inhibitors. 11,12 In line with these observations, it is suggested that Hsp90 regulates cell cycle checkpoints and DNA repair, 11-13 but the underlying mechanisms are poorly understood.Fanconi anemia (FA) is a genetically heterogeneous inherited disorder characterized by progressive bone marrow failure, cancer susceptibility, and cellular hypersensitivity to DNA cross-linkers such as mitomycin C (MMC). [14][15][16] Multiple FA proteins cooperate in a common biochemical pathway, termed the FA pathway, which is involved in cellular response to DNA damage. At least 8 FA proteins, specifically, FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL/PHF9, and FANCM, form a nuclear multiprotein complex (FA core complex), which is required for FANCD2 monoubiquitination in response to DNA damage. 17-32 DNA crosslinkers and replication inhibitors such as hydroxyurea (HU) are potent inducers of FANCD2 monoubiquitination. [14][15][16] The monoubiquitinated form of FANCD2 is targeted to the chromatin and participates in maintenance of genomic stability interacting with BRCA1 and BRCA2/FANCD1, at least in part, through homologydirected repair. 25,[33][34][35] FANCJ/BRIP1, previously identified as a BRCA1-interacting helicase, may function downstream of FANCD2 activation or independently of the FA pathway. [36][37][38] Previous studies suggested that nuclear levels of FANCA have profound effects on FA core complex formation. [17][18][19][20]24,25,27 Nuclear levels of FANCA are determined by protein synthesis, degradation, and nucleocytoplasmic shuttling mediated by a bipartite nuclear localization signal (NLS) and 3 leucine-rich nuclear export signals. 18,39,40 However, little is known about the regulatory mechanisms for intracellular turnover and trafficking of FANCA.In an attempt to elucidate the molecular m...