The 56 kDa androgen binding protein is an aldehyde dehydrogenase.

We have described a 56 kDa protein from genital skin fibroblasts that specifically binds androgen and that is generally not expressed in genital skin fibroblasts from patients with androgen insensitivity due to genetic defects of the androgen receptor. We have isolated a partial cDNA clone for the 56 kDa protein from an expression library of genital skin fibroblasts. In vitro translation of message selected with this clone faithfully produces the 56 kDa protein which can be immuneprecipitated with an anti-56 kDa antiserum. Northern blots probed with this clone show a 2.2 kb message, which parallels the expression of the 56 kDa protein. The sequence of this 998bp clone is identical to human liver aldehyde dehydrogenase 1, the cytoplasmic isoenzyme. On activity gels of genital skin fibroblast cytosol covalently labelled with androgen, aldehyde dehydrogenase activity comigrates with the single band labelled specifically with androgen. Thus, the 56 kDa androgen binding protein is an aldehyde dehydrogenase, which is prominently expressed in normal genital skin fibroblasts, but not in non-genital skin fibroblasts.     

A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-alpha but has no activity.

When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function.     

A 53 kDa protein binds to the negative regulatory region of JC virus early promoter.

Using gel retardation and photocrosslinking experiments, we have identified proteins of about 53 kDa in size in both rat glioma (C6) and cervical carcinoma (HeLa) cell extracts which bind to the negative regulatory region of JV virus (JCV) early promoter. The glial cell protein binds to its cognate promoter element with lesser affinity when compared to the protein present in HeLa cells. Further, these proteins interacted differentially to an oligonucleotide, containing the neighboring cis-acting domain, which is recognized by nuclear factor 1 (NF1). These findings suggest that the interactions of the 53 kDa HeLa protein may contribute to the negative regulation of JCV early promoter in HeLA cells.     

A eukaryotic nuclear protein of 130 kDa binds to a bacterial cAMP responsive element.

It has been known that one of the signal transduction mechanisms in Escherichia coli is mediated by cAMP which binds to the receptor protein (CAP), and that CAP complexed with cAMP facilitates gene expression by binding to the specific sequences. To identify a molecular mechanism in eukaryotes similar to a cAMP-mediated pathway in E. coli, the function of the CAP binding site of lac gene in E. coli and the protein(s) interacting with it were examined in a mammalian system. From transient expression studies of the fusion gene between the chloramphenicol acetyltransferase and lac genes, it was found that the lacCAP binding site could act as an enhancer activity on the SV40 promoter, and also as an additive enhancer activity to the SV40 enhancer in HeLa cells. However, the activity was not stimulated by cpt-cAMP (a highly stable analogue of cAMP) in HeLa cells, although it was induced in PC12 cells. These results suggest that a bacterial cAMP responsive element may function also in eukaryotes as a cis-acting element in a cell type dependent manner. Results from gel mobility shift assays showed that a protein(s) exists that specifically binds to the lacCAP binding site in eukaryotic nuclear extracts. As one of the proteins binding to the above site, we have identified a 130 kDa protein by using the Southwestern method. Although a function of the 130 kDa protein has not yet been understood, there is a possibility that the 130 kDa protein may play a role in the regulation of cAMP-dependent gene expression.     

Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.     

Binding of pepsinogen to the 78 kDa gastrin binding protein.

An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31+/-0.09 microgram/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2,17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathione-S-transferase fusion proteins in Escherichia coli, and purified on glutathione-agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119-157 and 199-219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9+/-1. 2 micrograms/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A.     

Selenite binding to carbon monoxide oxidase from Pseudomonas carboxydovorans. Selenium binds covalently to the protein and activates specifically the CO----methylene blue reaction

The CO----methylene blue and CO----dichlorophenol indophenol activities of carbon monoxide oxidase were specifically activated upon aerobic incubation with selenite, whereas the NADH----methylene blue activity was not altered. Fully active enzyme contained selenium, molybdenum, and flavin adenine dinucleotide in a 1:1:1 ratio. Selenium was covalently bound to the protein, probably between the sulfurs of half-cystine residues, and not a constituent of the molybdenum cofactor. The action of selenite was directed to the cytoplasmic species of carbon monoxide oxidase exclusively, whereas the CO----methylene blue activity of the membrane-bound enzyme remained unaffected.     

Prokaryotic calcium-binding protein of the calmodulin superfamily. Calcium binding to a Saccharopolyspora erythraea 20 kDa protein.

The EF-hand calcium-binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd(2+)-ion binding sites. This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd(2+)-ions. Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK1 = 8.0, pK2 = 9.0) are strongly cooperative, and one single site (pK3 = 7.5). Preliminary 1H NMR experiments indicate marked structural changes upon Ca(2+)-binding.     

Manganese binding to the 23 kDa extrinsic protein of Photosystem II

The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K(A)=10(-17) M(-1), was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.     

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.     

Bacteriophage phi X174 A protein cleaves single-stranded DNA and binds to it covalently through a tyrosyl-dAMP phosphodiester bond.

The phi X174 A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated end. To determine the nature of the covalent linkage and the amino acid involved, we used the A protein to cleave DNA synthesized in vitro with [alpha-32P]dATP to form the complex of A protein covalently linked to single-stranded DNA. The complex was then digested with DNase I, and the 32P-labeled A protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was treated extensively with trypsin, and the released peptide-oligonucleotide complexes were incubated with formic acid and diphenylamine (Burton reaction). The Burton reaction caused a transfer of the labeled phosphate from dAMP to the peptide. The labeled phosphopeptides were isolated and hydrolyzed, revealing a linkage of the phosphate to a tyrosine. These results indicate that the A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus by a tyrosyl-dAMP phosphodiester bond.     

Hsp56 mRNA in Paracentrotus lividus embryos binds to a mitochondrial protein

We previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it has a specific territorial distribution, both in normal and heat-shocked embryos, and that its amount increases after heat shock [Roccheri MC, Patti M, Agnello M, Gianguzza F, Carra E, Rinaldi AM. Localization of mitochondrial Hsp56 chaperonin during sea urchin development. Biochem Biophys Res Commun 2001;287:1093-98] and cadmium treatment [Roccheri MC, Agnello M, Boneventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004;321:80-7]. In this study, we looked at Hsp56 mRNA during normal development and under stress conditions. The messenger is almost constantly expressed at all stages of development and its amount is steadily increased in stressed embryos. Moreover, we found, using T1 RNase protection assay, that the most proximal region of the 3'-UTR of the Hsp56 mRNA binds a 40 kDa protein: this factor is more abundant in the mitochondrial extract and, more specifically, in the outer membrane of the organelle.     

A cellular 57 kDa protein binds to two regions of the internal translation initiation site of foot-and-mouth disease virus

A ribosome-associated 57 kDa protein from rabbit reticulocytes was linked to the internal translation initiation site of foot-and-mouth disease virus by mild UV-irradiation. Binding studies with different RNA fragments revealed that this protein interacts with two distinct sites within the translational control region. One site is located approximately 400 nucleotides upstream from the translational start codon and the second binding site could be confined to 60 nucleotides preceding this codon. Both sequences coincide with hairpin structures at the two opposite ends of a secondary structure model of the internal ribosomal entry site proposed by Pilipenko et al. [(1989) Nucleic Acids Res. 17, 5701-5711].     

Determinants for binding of a 40 kDa protein to the leaders of yeast mitochondrial mRNAs.

An abundant yeast mitochondrial 40 kDa protein (p40) binds with high specificity to the 5'-untranslated region of cytochrome c oxidase subunit II (COX2) mRNA. Using mobility shift and competition assays, we show here that purified p40 complexes with the leaders of all eight mitochondrial mRNAs of Saccharomyces cerevisiae. The location of the protein binding site on the different leaders is not conserved with respect to the AUG start codon. In vitro RNA footprint and deletion experiments have been used to define the p40-binding site on the leaders of COX1 and ATP9 mRNAs. Nucleotides at, and near, a single stranded region are protected or exposed for DEPC modification by binding of p40 to these leaders. Removal of this region from the COX1 messenger shows that it is essential for the protein-RNA interaction. While no obvious sequence similarity can be detected between the single stranded regions in different leaders, a nearby helical segment is conserved. A consensus model for p40-RNA interactions is presented and the possible biological function of p40 is discussed.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

Identification of a 67 kDa protein that binds specifically to the pre-rRNA primary processing site in a higher plant.

In radish pre-rRNA primary processing cleavage occurs at a UUUUCGCGC element (motif P) mapped in the 5'-external transcribed spacer (Delcasso-Tremousaygue et al., 1988). Significantly, motif P is part of a cluster of homologous elements including three UUUUCCGG elements (motifs A123) and a single UUUUGCCCC element (motif B). Here we used the EMSA to identify in radish extracts an RNA-binding activity, NF C, that specifically interacts with the pre-rRNA A123BP sequence. Using different RNA probes and competitors we show that NF C recognises a 38 base RNA sequence including the 3'-end of motif A3 and motifs B and P. NF C binds to poly U, but not to poly A, poly C or poly G. Therefore we used poly (U) Sepharose chromatography as a final step to obtain pure NF C fractions. These, analysed by SDS-PAGE, revealed two major polypeptides of 67 and 60 kDa. According to UV cross-linking analysis the 67 kDa polypeptide corresponds to NF C activity, while the 60 kDa species is a proteolysed form of this protein. We also showed that NF C is enriched in nuclear extracts. Based on its stringent RNA substrate specificity and its nuclear localisation we propose that NF C is involved in pre-rRNA primary processing in plants.     

Manganese binding to the 23 kDa extrinsic protein of Photosystem II

The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K_A = 10^− 17 M^− 1, was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.     

Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue of protein phosphatase 2A catalytic subunit in mammalian cells

Fostriecin is a phosphate monoester with excellent antitumor activity against mouse leukemia, and it is a potent inhibitor of protein phosphatase (PP) 2A. This compound has been predicted to covalently bind to the Cys269 residue of the PP2A catalytic subunit (PP2Ac) at the α,β-unsaturated lactone via a conjugate addition reaction. However, this binding has not yet been experimentally proven. To confirm such binding, we synthesized biotin-labeled fostriecin (bio-Fos), which has an inhibitory activity against the proliferation of mouse leukemia cells. We showed that fostriecin directly binds to PP2Ac in HeLa S3 cells by pull-down assays using bio-Fos. Moreover, we directly demonstrated that fostriecin covalently binds to the Cys269 residue of PP2Ac by matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis. From these results, the inhibitory mechanism of fostriecin on PP2A activity is discussed.