Large-scale analysis of the human ubiquitin-related proteome

Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin-conjugated and ubiquitin-associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2-D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp-D (ubiquitin-related proteome under the denaturing condition) and comprised ubiquitin-conjugated molecules, whereas 325 proteins (49%) were classified as Urp-N (ubiquitin-related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp-D and Urp-N. Many ribosomal subunits were detected in the Urp-D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin-related cellular functions.     

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2).

Abstract

LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.

     

Protracted Administration of L-Asparaginase in Maintenance Phase Is the Risk Factor for Hyperglycemia in Older Patients with Pediatric Acute Lymphoblastic Leukemia

Although L-asparaginase related hyperglycemia is well known adverse event, it is not studied whether the profile of this adverse event is affected by intensification of L-asparaginase administration. Here, we analyzed the profile of L-asparaginase related hyperglycemia in a 1,176 patients with pediatric acute lymphoblastic leukemia treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol using protracted L-asparaginase administration in maintenance phase. We determined that a total of 75 L-asparaginase related hyperglycemia events occurred in 69 patients. Although 17 events (17/1176, 1.4%) developed in induction phase, which was lower incidence than those (10–15%) in previous reports, 45 events developed during the maintenance phase with protracted L-asparaginase administration. Multivariate analysis showed that older age at onset (≥10 years) was a sole independent risk factor for L-asparaginase-related hyperglycemia (P<0.01), especially in maintenance phase. Contrary to the previous reports, obesity was not associated with L-asparaginase-related hyperglycemia. These findings suggest that protracted administration of L-asparaginase is the risk factor for hyperglycemia when treating adolescent and young adult acute lymphoblastic leukemia patients.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Peptide condensation activity of a neutral protease from Vibrio sp. T1800 (Vimelysin)

Condensation of Cbz-Asp and PheOMe catalyzed by a neutral protease from Vibrio sp. T1800 (Vimelysin: VLN) was studied. VLN showed a relatively higher catalytic activity of condensation and an apparently larger yield after 3 h or 24 h, in comparison with thermolysin (TLN), especially at lower pH and temperatures.VLN showed higher solvent-tolerance than TLN. TLhe apparent highest yield (25%) was obtained in 30% DMSO by using VLN; under similar conditions, TLN gave only about a half of this value. The rate of the condensation reaction per mole of enzyme (v/[E](o)) in DMSO 50% at 37 degrees C and pH 6.5 was 0.16 s(-1) for VLN and 0.047 s(-1) for TLN. In 30% ethanol VLN showed more than three-fold peptide yield than TLN after 5 h reaction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 387-390, 1997.     

PAL31 expression in rat trophoblast giant cells

PAL31 is a proliferation-related acidic nuclear protein that belongs to the leucine-rich protein family and is expressed cell-cycle-dependently. Trophoblasts differentiate into the trophoblast giant cells (TGCs) through the unusual type of cell cycle, namely endoreduplication. In the present study, we investigated the spatiotemporal pattern of PAL31 expression in rat placenta and Rcho-1 cell line. The PAL31 mRNA concentration varied in different areas of the placenta, and was barely detectable in the TGC layer. In Rcho-1 cells, although the level of PAL31 mRNA decreased dramatically during differentiation, PAL31 was detected even after differentiation. The site of intranuclear localization of PAL31 mostly overlapped with that of PCNA in the undifferentiated Rcho-1 cells, while they were not overlapped in differentiated cells. Thus, the subcellular localization of PAL31 in Rcho-1 cells significantly changed, and loss of cell cycle dependency after differentiation was noted. PAL31 is suggested to play a role in the endoreduplication distinct from the usual DNA duplication.     

Novel kinetic analysis of enzymatic dipeptide synthesis: effect of pH and substrates on thermolysin catalysis.

The point of maximum activity is specific to a particular substrate-enzyme system but may vary with different substrates and the same enzyme. The specificity of enzymes has, however, been generally reported only at their "optimal" pH. In this article, we introduce the Michaelis-Menten equation taking pH into account, and apply it to the pH-activity profile of the thermolysin-catalyzed dipeptide synthesis. It has been reported to date that the pH-activity profile of thermolysin follows a bell-shaped curve with a maximal activity at or near pH 7.0. The profiles obtained in this study, however, indicated that the optimal pH varied from 5.8 (for F-AspPheOMe) to 7.3 (for Z-ArgPheOMe), and the order of thermolysin activity was greatly dependent on the pH of reaction media. We have succeeded in evaluating the substrates-induced change of the dissociation states of the active site of thermolysin using the hydrophobicity of substrates. We have obtained apparent kinetic parameters which are independent of the pH of reaction media. The apparent specificity of thermolysin which were independent of pH of the reaction media was in order L-Leu > L-Asp > L-Arg > L-Ala > L-Gly > L-Val and Z > Boc = F at P1 and P2 positions, respectively.     

Crystal structure of tobacco necrosis virus at 2.25 A resolution

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.