Exon Elongation Added Intrinsically Disordered Regions to the Encoded Proteins and Facilitated the Emergence of the Last Eukaryotic Common Ancestor.

Most prokaryotic proteins consist of a single structural domain (SD) with little intrinsically disordered regions (IDRs) that by themselves do not adopt stable structures, whereas the typical eukaryotic protein comprises multiple SDs and IDRs. How eukaryotic proteins evolved to differ from prokaryotic proteins has not been fully elucidated. Here, we found that the longer the internal exons are, the more frequently they encode IDRs in eight eukaryotes including vertebrates, invertebrates, a fungus, and plants. Based on this observation, we propose the "small bang" model from the proteomic viewpoint: the protoeukaryotic genes had no introns and mostly encoded one SD each, but a majority of them were subsequently divided into multiple exons (step 1). Many exons unconstrained by SDs elongated to encode IDRs (step 2). The elongated exons encoding IDRs frequently facilitated the acquisition of multiple SDs to make the last common ancestor of eukaryotes (step 3). One prediction of the model is that long internal exons are mostly unconstrained exons. Analytical results of the eight eukaryotes are consistent with this prediction. In support of the model, we identified cases of internal exons that elongated after the rat-mouse divergence and discovered that the expanded sections are mostly in unconstrained exons and preferentially encode IDRs. The model also predicts that SDs followed by long internal exons tend to have other SDs downstream. This prediction was also verified in all the eukaryotic species analyzed. Our model accounts for the dichotomy between prokaryotic and eukaryotic proteins and proposes a selective advantage conferred by IDRs.

Extracellular HSPA5 is autocrinally involved in the regulation of neuronal process elongation.

The molecular mechanisms by which neuronal processes grow are extremely complicated, involving fine-tuned regulation of extracellular and intracellular signals. It remains to be elucidated which molecules are contained in the regulation. Herein, we report for the first time that heat shock protein family A member 5 (HSPA5, also called immunoglobulin heavy chain binding endoplasmic reticulum [ER] protein [BiP]) is secreted from mouse primary dorsal neuronal ganglion (DRG) cells or neuronal cell line N1E-115, a frequently used neuronal differentiation model. Supporting these results, HSPA5 protein was co-localized not only with ER antigen KDEL but also with intracellular vesicles such as Rab11-positive secretory vesicles. Unexpectedly, addition of HSPA5 inhibited elongation of neuronal processes, whereas neutralization of extracellular HSPA5 with the antibodies elongated processes, characterizing extracellular HSPA5 as a negative regulator of neuronal differentiation. Treatment of cells with neutralizing antibodies for low-density lipoprotein receptor (LDLR) did not have significant effects on process elongation, whereas LDLR-related protein 1 (LRP1) antibodies promoted differentiation, implying that LRP1 may act as a receptor candidate for HSPA5. Interestingly, extracellular HSPA5 was greatly decreased following treatment with tunicamycin, an ER stress inducer, illustrating that the ability to form neuronal processes could be preserved, even under stress. These results suggest that neuronal HSPA5 itself is secreted to contribute to inhibitory effects on neuronal cell morphological differentiation and can be included on the list of extracellular signaling molecules negatively controlling differentiation.

Nuclear grade and comedo necrosis of ductal carcinoma in situ as histopathological eligible criteria for the Japan Clinical Oncology Group 1505 trial: an interobserver agreement study.

OBJECTIVE: The Japan Clinical Oncology Group 1505 trial is a single-arm multicentre prospective study that examined the possibility of non-surgical follow-up with endocrine therapy for patients with low-grade ductal carcinoma in situ. In that study, the eligible criteria included histopathological findings comprising low to intermediate nuclear grade and absence of comedo necrosis, and cases were entered according to the local histopathological diagnosis. Nuclear grade is largely based on the Consensus Conference criteria (1997), whereas comedo necrosis is judged according to the Rosen's criteria (2017). The purpose of this study was to standardize and examine the interobserver agreement levels of these histopathological criteria amongst the participating pathologists. METHODS: We held slide conferences, where photomicrographs of haematoxylin-eosin-stained slides from 68 patients with ductal carcinoma in situ were presented using PowerPoint. The nuclear grade and comedo necrosis statuses individually judged by the pathologists were analysed using κ statistics. RESULTS: In the first and second sessions, where 22 cases each were presented, the interobserver agreement levels of nuclear grade whether low/intermediate grade or high grade were moderate amongst 29 and 24 participating pathologists, respectively (κ = 0.595 and 0.519, respectively). In the third session where 24 cases were presented, interobserver agreement levels of comedo necrosis or non-comedo necrosis were substantial amongst 25 participating pathologists (κ = 0.753). CONCLUSION: Although the concordance rates in nuclear grade or comedo necrosis were not high in a few of the cases, we believe that these results could provide a rationale for employing the present criteria of nuclear grade and comedo necrosis in the clinical study of ductal carcinoma in situ.

Crumbs3 is a critical factor that regulates invasion and metastasis of colon adenocarcinoma via the specific interaction with FGFR1.

Epithelial cell polarity regulator Crumbs3 (Crb3), a mammalian homolog within the Drosophila Crb gene family, was initially identified as an essential embryonic development factor. It is recently implicated in tumor suppression, though its specific functions are controversial. We here demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. Crb3 centrality to tumor migration was supported by strong expression at invasive front and metastatic foci of colonic adenocarcinoma of the patient tissues. Accordingly, two different Crb3-knockout (KO) lines, Crb3-KO (Crb3 -/-) DLD-1 and Crb3-KO WiDr from human colonic adenocarcinomas, were generated by the CRISPR-Cas9 system. Crb3-KO DLD-1 cells exhibited loss of cellular mobility in vitro and dramatic suppression of liver metastases in vivo in contrast to the wild type of DLD-1. Unlike DLD-1, Crb3-KO WiDr mobility and metastasis were unaffected, which were similar to wild-type WiDr. Proteome analysis of Crb3-coimmunopreciptated proteins identified different respective fibroblast growth factor receptor (FGFR) isotypes specifically bound to Crb3 isoform a through their intracellular domain. In DLD-1, Crb3 showed membranous localization of FGFR1 leading to its functional activation, whereas Crb3 bound to cytoplasmic FGFR4 in WiDr without FGFR1 expression, leading to cellular growth. Correlative expression between Crb3 and FGFR1 was consistently detected in primary and metastatic colorectal cancer patient tissues. Taking these together, Crb3 critically accelerates cell migration, namely invasion and metastasis of human colon cancers, through specific interaction to FGFR1 on colon cancer cells.

SMAD4 alteration associates with invasive-front pathological markers and poor prognosis in colorectal cancer.

AIMS: SMAD4 acts as a tumour suppressor, and the loss of SMAD4 is associated with poor prognosis in colorectal cancer (CRC) patients. Although next-generation sequencing (NGS) enabled us to detect numerous genetic alterations in a single assay, the clinical significance of SMAD4 alteration detected with NGS has not been fully investigated. The aim of this study was to evaluate the clinicopathological characteristics and clinical significance of SMAD4 alteration detected with NGS in CRC. METHODS AND RESULTS: We retrospectively investigated 201 patients with stage I-IV CRC, by using a 415-gene panel. To analyse the relationship between SMAD4 alteration and other clinicopathological characteristics, we evaluated clinicopathological variables, including invasive-front pathological markers: tumour budding, poorly differentiated cluster, and Crohn-like lymphoid reaction. Fifty-six patients (28%) had SMAD4 alteration: 24 and 32 patients had SMAD4 mutation and deletion, respectively. SMAD4 alteration was significantly associated with T category (P = 0.027), N category (P = 0.037), M category (P = 0.028), and invasive-front pathological markers, such as poorly differentiated cluster grade 3 (P = 0.020) and absence of Crohn-like lymphoid reaction (P = 0.004). Immunohistochemistry revealed that SMAD4 alteration was significantly associated with loss of SMAD4 (P = 0.023). In 90 patients with stage I-III disease, SMAD4 alteration was significantly associated with poor prognosis for relapse-free and overall survival (P = 0.047; P = 0.022, respectively). Conversely, in 111 patients with stage IV disease, SMAD4 alteration was not significantly associated with overall survival. CONCLUSION: SMAD4 alteration is associated with invasive-front pathological markers and poor prognosis in stage I-III CRC patients.

Dystonia-4 (DYT4)-associated TUBB4A mutants exhibit disorganized microtubule networks and inhibit neuronal process growth.

Dystonia-1 (DYT1) is an autosomal dominant early-onset torsion form of dystonia, a neurological disease affecting movement. DYT1 is the prototypic hereditary dystonia and is caused by the mutation of the tor1a gene. The gene product has chaperone functions important for the control of protein folding and stability. Dystonia-4 (DYT4) is another autosomal dominant dystonia that is characterized by onset in the second to third decade of progressive laryngeal dysphonia. DYT4 is associated with the mutation of the tubb4a gene, although it remains to be understood how disease-associated mutation affects biochemical as well as cell biological properties of the gene product as the microtubule component (a tubulin beta subunit). Herein we demonstrate that DYT4-associated TUBB4A missense mutants (Arg2-to-Gly or Ala271-to-Thr) form disorganized tubulin networks in cells. Transfected mutants are indeed expressed in cytoplasmic regions, as observed in wild-type transfectants. However, mutant proteins do not exhibit typical radial tubulin networks. Rather, they have diminished ability to interact with tubulin alpha subunits. Processes do not form in sufficient amounts in cells of the N1E-115 neuronal cell line expressing each of these mutants as compared to parental cells. Together, DYT4-associated TUBB4A mutants themselves form aberrant tubulin networks and inhibit neuronal process growth, possibly explaining progress through the pathological states at cellular levels.

Pull down assay for GTP-bound form of Sar1a reveals its activation during morphological differentiation.

The intracellular molecular transport system is a basic and general cellular mechanism that is regulated by an array of signaling molecules. Sar1 small GTPases are molecules that play a key role in controlling vehicle transport between the endoplasmic reticulum (ER) and Golgi bodies. Like other small GTPases, the activities of Sar1a depend on their guanine-nucleotide-binding states, which are regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the well-known function of mammalian Sar1 in the intracellular transport system, little is known about when and how Sar1 is activated during cell morphological changes. Here we show that the C-terminal, but not the N-terminal, regions of Sec23A and Sec23B, the effector proteins of Sar1a, specifically bind to the active, GTP-bound form of Sar1a. An affinity precipitation (pull-down) assay using a recombinant C-terminal region of Sec23B reveals that Sar1a is activated following differentiation in neuronal cell lines. In neuronal N1E-115 cells, GTP-bound Sar1a is increased when cells elongate neuronal processes. Similar results are observed in morphological differentiation in oligodendroglial FBD-102b cells. Additionally, prolactin regulatory element binding (PREB), the GEF for Sar1 (Sar1 activator), increases the binding ability to the nucleotide-free form of Sar1a when morphological differentiation occurs. Nucleotide-free small GTPases preferentially interact with the cognate, active GEFs. These results provide evidence that using previously unreported pull down assays reveals that Sar1 and PREB are upregulated following the induction of morphological differentiation, suggesting the potential role of signaling through Sar1a during morphological differentiation.

Codon usage is less optimized in eukaryotic gene segments encoding intrinsically disordered regions than in those encoding structural domains.

Codon usage tends to be optimized in highly expressed genes. A plausible explanation for this phenomenon is that translational accuracy is increased in highly expressed genes with infrequent use of rare codons. Besides structural domains (SDs), eukaryotic proteins generally have intrinsically disordered regions (IDRs) that by themselves do not assume unique three-dimensional structures. As IDRs are free from structural constraint, they can probably accommodate more translational errors than SDs can. Thus, codon usage in IDRs is likely to be less optimized than that in SDs. Codon usage in all the genes of seven eukaryotes was examined in terms of both tRNA adaptation index and codon adaptation index. Different amino acid compositions in different protein regions were taken into account in calculating expected adaptation indices, to which observed indices were compared. Codon usage is less optimized in gene regions encoding IDRs than in those corresponding to SDs. The finding does not depend on whether IDRs are located at the N-terminus, in the middle, or at the C-terminus of proteins. Furthermore, the observation remains unchanged in two different algorithms used to predict IDRs in proteins. The result is consistent with the idea that IDRs tolerate more translational errors than SDs.

Both Intrinsically Disordered Regions and Structural Domains Evolve Rapidly in Immune-Related Mammalian Proteins.

Eukaryotic proteins consist of structural domains (SDs) and intrinsically disordered regions (IDRs), i.e., regions that by themselves do not assume unique three-dimensional structures. IDRs are generally subject to less constraint and evolve more rapidly than SDs. Proteins with a lower number of protein-to-protein interactions (PPIs) are also less constrained and tend to evolve fast. Extracellular proteins of mammals, especially immune-related extracellular proteins, on average have relatively high evolution rates. This article aims to examine if a high evolution rate in IDRs or that in SDs accounts for the rapid evolution of extracellular proteins. To this end, we classified eukaryotic proteins based on their cellular localizations and analyzed them. Moreover, we divided proteins into SDs and IDRs and calculated the respective evolution rate. Fractional IDR content is positively correlated with evolution rate. For their fractional IDR content, immune-related extracellular proteins show an aberrantly high evolution rate. IDRs evolve more rapidly than SDs in most subcellular localizations. In extracellular proteins, however, the difference is diminished. For immune-related proteins in mammals in particular, the evolution rates in SDs come close to those in IDRs. Thus high evolution rates in both IDRs and SDs account for the rapid evolution of immune-related proteins.

Formalin-fixed paraffin-embedded sample conditions for deep next generation sequencing.

INTRODUCTION: Precision medicine is only possible in oncology practice if targetable genes in fragmented DNA, such as DNA from formalin-fixed paraffin-embedded (FFPE) samples, can be sequenced using next generation sequencing (NGS). The aim of this study was to examine the quality and quantity of DNA from FFPE cancerous tissue samples from surgically resected and biopsy specimens. METHODS: DNA was extracted from unstained FFPE tissue sections prepared from surgically resected specimens of breast, colorectal and gastric cancer, and biopsy specimens of breast cancer. A total quantity of DNA ≥60 ng from a sample was considered adequate for NGS. The DNA quality was assessed by Q-ratios, with a Q-ratio >0.1 considered sufficient for NGS. RESULTS: The Q-ratio for DNA from FFPE tissue processed with neutral-buffered formalin was significantly better than that processed with unbuffered formalin. All Q-ratios for DNA from breast, colorectal and gastric cancer samples indicated DNA levels sufficient for NGS. DNA extracted from gastric cancer FFPE samples prepared within the last 7 years is suitable for NGS analysis, whereas those older than 7 years may not be suitable. Our data suggested that adequate amounts of DNA can be extracted from FFPE samples, not only of surgically resected tissue but also of biopsy specimens. CONCLUSIONS: The type of formalin used for fixation and the time since FFPE sample preparation affect DNA quality. Sufficient amounts of DNA can be extracted from FFPE samples of both surgically resected and biopsy tissue, thus expanding the potential diagnostic uses of NGS in a clinical setting.

[Long-Term Complete Response in Postoperative Multiple Lung and Mediastinal Lymph Node Metastases of Esophageal Cancer Using Systemic Chemotherapy with DCF Treatment - A Case Report].

A 59-year-old man was diagnosed with advanced thoracic esophageal cancer.He underwent neoadjuvant chemotherapy with cisplatin(CDDP), 5-fluorouracil(5-FU)(CF)followed by transthoracic esophagectomy with three-field lymphadenectomy. Histopathological examination revealed that the tumor was poorly differentiated squamous cell carcinoma invading the adventitia with 6 regional lymph node metastases.Four months after surgery, follow-up thoracic computed tomography (CT)showed multiple lung and mediastinal lymph node metastases.The patient's general condition was favorable, and he underwent systemic chemotherapy with docetaxel, CDDP, 5-FU(DCF).After 2 courses of DCF, chest CT revealed that the lung and mediastinal lymph node metastases had markedly decreased in size to the point of being unmeasurable.After receiving additional chemotherapy consisting of 2 courses of DCF and 8 courses of monthly docetaxel, the patient has been followed up without treatment.No tumor re-recurrence has occurred in the 6 years and 8 months since the first recurrence.In cases of recurrent or unresectable esophageal cancer, the rate of clinical response is reported to be higher for DCF than for CF. DCF is a tolerable regimen, even for postoperative patients, provided that monitoring is conducted for severe adverse events. In patients whose general conditions are favorable, DCF should be considered as a treatment option for recurrent esophageal cancer.

New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.

Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth ß-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC-CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.

The clinical significance of potentially curative resection for gastric cancer following the clearance of free cancer cells in the peritoneal cavity by induction chemotherapy.

PURPOSE: The aim of this study was to evaluate the significance of the conversion of the peritoneal cytology from positive to negative following induction chemotherapy in relation to the prognosis after subsequent resection for gastric cancer. METHODS: This retrospective study was conducted using a prospectively maintained database. A total of 47 patients with free cancer cells in the peritoneal cavity, as evaluated by staging laparoscopy, but no other evidence of distant metastasis, who underwent induction chemotherapy followed by surgery were enrolled in the study. Then, the clinicopathological factors and survival in the study subjects were assessed. RESULTS: The median survival time and 5-year overall survival rate of the 47 study participants were 20.4 months and 25.0 %, respectively. In 23 of the patients, the peritoneal cytology converted from positive to negative after the induction chemotherapy, and a microscopically margin-negative gastrectomy was performed. The median survival time of 30.4 months and the 5-year survival rate of 34.6 % of these patients was significantly more favorable than the corresponding values of 15.0 months and 17.6 % in the patients who had persistently positive cytology (P = 0.03). CONCLUSION: Based on our findings, the clearance of free cancer cells in the peritoneal cavity by induction chemotherapy improves the prognosis of patients following subsequent gastrectomy.

Splicing factor 2-associated protein p32 participates in ribosome biogenesis by regulating the binding of Nop52 and fibrillarin to preribosome particles.

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52.

The Autophagy Database: an all-inclusive information resource on autophagy that provides nourishment for research.

Autophagy is a process of self-digestion generally observed in eukaryotes and has been shown to play crucial roles for survival under starvation and removal of deleterious substances. Despite great advances that have been made, many problems in mechanisms of autophagy remain unsolved. As a large number of autophagy-related proteins are identified in each species, a database that collects data, identifies their homologs in other species and makes them available will contribute to research advancement. As no such resources exist, we built the Autophagy database (http://tp-apg.genes.nig.ac.jp/autophagy) to provide basics, up-to-date information on relevant literature, and a list of autophagy-related proteins and their homologs in 41 eukaryotes. From the database, the user can search for proteins by keywords or sequences to obtain a wealth of data including functional and structural information and find possible functional homologs of proteins whose functions have been demonstrated in other species. As proteins that bind the phospholipid, phosphatidyl inositol 3-phosphate (PI3P) are essential for autophagy to proceed, we carried out an original analysis to identify probable PI3P-binding proteins, and made the list available from the database. The database is expected to give impetus to further research on autophagy by providing basic and specialized data on the subject.

Routine clinical use of the one-step nucleic acid amplification assay for detection of sentinel lymph node metastases in breast cancer patients: results of a multicenter study in Japan.

BACKGROUND: The objective of this study was to confirm, by means of a multicenter study conducted in Japan, the reliability and usefulness of the one-step nucleic acid amplification (OSNA) assay in routine clinical use for sentinel lymph node biopsy (SLNB) of breast cancer patients. METHODS: Patients with Tis-T2N0M0 breast cancer who underwent SLNB before systemic chemotherapy comprised the study cohort. A whole sentinel lymph node (SLN) was examined intraoperatively with the OSNA assay except for a 1-mm-thick, central slice of the lymph node, which underwent pathologic examination after the operation. For patients who underwent axillary dissection, non-SLNs were examined with routine pathologic examination. RESULTS: In total, 417 SLNBs from 413 patients were analyzed. SLN metastases were detected with greater sensitivity by the OSNA assay than by pathologic examination (22.5% vs 15.8%; P < .001), as expected from the difference in size of the specimens examined. Patients who had SLN metastases assessed with the OSNA assay proved to harbor non-SLN metastases with an overall risk ratio of 33.7%. The risk of non-SLN metastasis was significantly lower for patients who had positive SLNs assessed as OSNA+ than for those who had SLNs assessed as OSNA++ (17.6% vs 44%; P = .012). CONCLUSIONS: The OSNA assay can be used for routine clinical SLNB, and its assessment for volume of metastasis may be a powerful predictive factor for non-SLN metastasis. Further studies with more patients are needed to confirm the usefulness of this assay for selection in the clinical setting of patients who do not need axillary dissection.

The adaptor SH2B1 and the phosphatase PTP4A1 regulate the phosphorylation of cytohesin-2 in myelinating Schwann cells in mice.

Mature myelin sheaths insulate axons to increase nerve conduction velocity and protect nerve fibers from stress and physical injury. In the peripheral nervous system, the myelin sheath is produced by Schwann cells. The guanine-nucleotide exchange factor cytohesin-2 activates the protein Arf6 to promote Schwann cell myelination. Here, we investigated the regulation of cytohesin-2 and found that the phosphorylation status of Tyr381 in cytohesin-2 is central to Schwann cell myelination. Knockin mice with a nonphosphorylatable Y381F mutation in cytohesin-2 exhibited reduced myelin thickness and decreased Arf6 activity in sciatic nerve tissue. In HEK293T cells, cytohesin-2 was dephosphorylated at Tyr381 by the protein tyrosine phosphatase PTP4A1, whereas phosphorylation at this site was maintained by interaction with the adaptor protein SH2B1. Schwann cell-specific knockdown of PTP4A1 in mice increased cytohesin-2 phosphorylation and myelin thickness. Conversely, Schwann cell-specific loss of SH2B1 resulted in reduced myelin thickness and decreased cytohesin-2 phosphorylation. Thus, a signaling unit centered on cytohesin-2-with SH2B1 as a positive regulator and PTP4A1 as a negative regulator-controls Schwann cell myelination in the peripheral nervous system.

Binary classification of protein molecules into intrinsically disordered and ordered segments.

BACKGROUND: Although structural domains in proteins (SDs) are important, half of the regions in the human proteome are currently left with no SD assignments. These unassigned regions consist not only of novel SDs, but also of intrinsically disordered (ID) regions since proteins, especially those in eukaryotes, generally contain a significant fraction of ID regions. As ID regions can be inferred from amino acid sequences, a method that combines SD and ID region assignments can determine the fractions of SDs and ID regions in any proteome. RESULTS: In contrast to other available ID prediction programs that merely identify likely ID regions, the DICHOT system we previously developed classifies the entire protein sequence into SDs and ID regions. Application of DICHOT to the human proteome revealed that residue-wise ID regions constitute 35%, SDs with similarity to PDB structures comprise 52%, while SDs with no similarity to PDB structures account for the remaining 13%. The last group consists of novel structural domains, termed cryptic domains, which serve as good targets of structural genomics. The DICHOT method applied to the proteomes of other model organisms indicated that eukaryotes generally have high ID contents, while prokaryotes do not. In human proteins, ID contents differ among subcellular localizations: nuclear proteins had the highest residue-wise ID fraction (47%), while mitochondrial proteins exhibited the lowest (13%). Phosphorylation and O-linked glycosylation sites were found to be located preferentially in ID regions. As O-linked glycans are attached to residues in the extracellular regions of proteins, the modification is likely to protect the ID regions from proteolytic cleavage in the extracellular environment. Alternative splicing events tend to occur more frequently in ID regions. We interpret this as evidence that natural selection is operating at the protein level in alternative splicing. CONCLUSIONS: We classified entire regions of proteins into the two categories, SDs and ID regions and thereby obtained various kinds of complete genome-wide statistics. The results of the present study are important basic information for understanding protein structural architectures and have been made publicly available at http://spock.genes.nig.ac.jp/~genome/DICHOT.

Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage.

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.