p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.     

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).     

Purification and Characterization of Cadmium-Binding Protein from Unicelluar Alga Chlorella sorokinian

The unicellular green alga Chlorella sorokiniana ANA9 is highly resistant to heavy metals, and its metal-binding proteins are induced in the presence of cadmium. A novel cadmium-binding protein in C. sorokiniana cultured in 100 mg/l cadmium ions for 4 days was isolated and characterized. The crude protein extract was obtained by cell disruption and partly purified by ammonium sulfate precipitation. After purification by anion-exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, the protein was further purified by gel filtration with Sephacryl S-100, followed by Sephadex G-75. The molecular weight of the purified protein was determined to be 11.5 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cadmium binding capacity of the purified protein was 119 μg/mg. The involvement of thiol coordination in metal-ion binding was confirmed by measuring the ultraviolet spectrum. This article is the first to describe the metallothionein-like cadmium-binding protein from Chlorella species, the expression of which is induced by cadmium exposure.     

Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.     

Solution structure of the antifreeze-like domain of human sialic acid synthase

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.     

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.     

Micro-analysis of Amino Acids with Fluorescein-isothiocyanate

The separation and identification of fluorescein-thiocarbarnyl (FTC-) amino acid II were accomplished by one- and two-dimensional thin-layer chromatography. The authentic samples for identification of amino acids were synthesized with fluorescein-isothiocyanate II (FITC II) and 21 amino acids. These FTC-amino acids were studied spectrometrically.

For quantitative estimation of FTC-amino acids, the fluoroscopy was used. It was found that the fluorescence intensity was proportional to the concentration of FTC-amino acids in 2 pmole/ml to 20 nmole/ml range. Recovery of FTC-derivatives on silica gel plate was about 80%.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Isolation and characterization of cytosolic and membrane-bound deubiquitinylating enzymes from bovine brain.

The deubiquitinylating enzymes (DUBs), that release free ubiquitin (Ub) from its precursors or ubiquitinylated proteins, are known to comprise of a large protein family in eukaryotes, but those in mammalian tissues remain largely unknown. Here we report the existence of unexpectedly large species of DUBs in both soluble and membrane-bound fractions of bovine brain, based on their ability to cleave (125)I-labeled Ub-fused alphaNH-MHISPPEPESEEEEEHYC (designated as Ub-PESTc). Two cytosolic enzymes, tentatively called sDUB-1 and sDUB-2, with molecular masses of about 30 kDa were purified to near homogeneity by Ub-Sepharose affinity chromatography. sDUB-1 and sDUB-2 corresponded to UCH-L3 and UCH-L1/PGP 9.5, respectively. Intriguingly, the particulate fraction of the brain homogenate was found to also contain strong activities against (125)I-Ub-PESTc, which can be solubilized by treatment with 5% n-heptyl-beta-D-thioglucoside and 1% Nonidet P-40, but not by washing with 1 M NaCl. From the solubilized material, two new 30-kDa, membranous DUBs (called mDUB-1 and mDUB-2) were purified to apparent homogeneity by Ub-Sepharose chromatography. Two other Ub-aldehyde sensitive DUBs, designated as mDUB-3 and mDUB-4, were also partially purified by conventional chromatographic operations. These mDUBs differed from each other in substrate specificity and exhibited different characteristics from the sDUBs, revealing that they are a new type of membrane-bound DUB. These results indicate the presence of divergent DUBs in mammalian brain, which may contribute to regulation of numerous pivotal cellular functions mediated by the covalent modification of Ub.     

Autocrine induction of substance P mRNA and peptide in cultured normal human keratinocytes.

In this study, we have demonstrated that normal cultured keratinocytes (KCs) could generate significant endogenous substance P (SP) in a dose- and time-dependent response to exogenous SP by sensitive ELISA assay and express preprotachinin-a mRNA by RT-PCR and Southern blotting. We performed immunohistochemical analysis to confirm the presence of SP in cultured keratinocytes. In contrast, adrenaline, acetylcholine, histamine and CGRP induced only low amount of SP from cultured normal human KCs. This is the first report that SP can be induced by skin epithelial cells in response to exogenous SP and KC derived SP might play an important role in induction and acceleration of certain cutaneous diseases.