Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes.

The effects of an affinity-purified polyclonal antibody to Artemia salina ribosomal protein L5 on protein synthesis in vitro were examined. The antibody interacted with 60 S subunits more strongly than with 80 S ribosomes, and inhibited reassociation of ribosomal subunits to some extent at 5 mM-Mg2+ but not at 10 mM. Polyphenylalanine synthesis in vitro at 10 mM-Mg2+ was significantly inhibited, especially when the antibody was first preincubated with 60 S subunits prior to the assay. The incorporation of amino acid directed by globin mRNA was inhibited only when the preincubation with 60 S subunits was carried out. On the other hand, no effect was detected on elongation factor 2- and 60 S subunit-dependent uncoupled GTPase activity. These results suggest that L5 is probably located at or near the subunit interface and may play an important role in protein synthesis.     

Essential structural features of acetogenins: role of hydroxy groups adjacent to the bis-THF rings

The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins. To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues which lack either or both of the hydroxy groups, and investigated their inhibitory activities with bovine heart mitochondrial complex I. Our results indicate that the presence of either of the two hydroxy groups sufficiently sustains a potent inhibitory effect.     

Dynamic function of the alkyl spacer of acetogenins in their inhibitory action with mitochondrial complex I (NADH-ubiquinone oxidoreductase)

Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

A liver-specific chromatin protein binding to the 5' far upstream region of the rat serum albumin gene

Chromatin proteins which were extracted with 0.3 M NaCl from rat liver, brain, and kidney nuclei were examined by the protein blotting technique for their ability to bind to the 5' upstream regions of the rat serum albumin gene. A 110-kDa protein from liver nuclei bound specifically to the most upstream fragment (between approximately equal to -7.3 kbp and -2.0 kbp from the cap site) of the cloned albumin genomic DNA, whereas no proteins from kidney and brain bound to this fragment. It is possible that the 110-kDa protein is concerned with the tissue-specific expression of the albumin gene.     

A comparative study on 40S ribosomal proteins of Artemia salina and rat liver: micro analysis of amino acid composition by high-performance liquid chromatography

The amino acid compositions of 24 proteins of 40S ribosomal subunits of Artemia salina cysts were determined and compared with those of rat liver. The basic proteins of A. salina 40S ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and extracted with 70% formic acid. Samples were freed from contaminants by gel-filtration through a high-performance liquid chromatography column. Amino acid compositions were determined for individual proteins by pre-column derivatization with N,N-dimethylaminoazobenzenesulfonyl chloride followed by reverse phase high-performance liquid chromatography. The similarity of amino acid compositions between A. salina and rat liver 40S ribosomal proteins was evaluated by the method of Cornish-Bowden (Cornish-Bowden, A. (1980) Anal. Biochem. 105, 233-238), and possible relationships between A. salina and rat were detected for 16 protein species (S2, S3, S4, S6, S7, S8, S15a, S16, S17, and S18, strongly related and S14, S15, S20, S23, S24, and S26, weakly related), indicating a conservative nature of eukaryotic ribosomal proteins.     

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.     

Age- and α-Synuclein-Dependent Degeneration of Dopamine and Noradrenaline Neurons in the Annual Killifish Nothobranchius furzeri

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Synthesis and inhibitory action of novel acetogenin mimics with bovine heart mitochondrial complex I

Studies on the inhibition mechanism of acetogenins, the most potent inhibitors of complex I, are useful to elucidate the structural and functional features of the terminal electron-transfer step of this enzyme. We synthesized acetogenin mimics that possess two alkyl tails without a gamma-lactone ring, named Deltalac-acetogenin, and examined their inhibitory action on bovine heart mitochondrial complex I. Unexpectedly, the Deltalac-acetogenin carrying two n-undecanyl groups (compound 3) elicited very potent inhibition comparable to that of bullatacin. The inhibitory potency of compound 3 markedly decreased with shortening the length of either or both alkyl tails, indicating that symmetric as well as hydrophobic properties of the inhibitor are important for the inhibition. Both acetylation and deoxygenation of either or both of two OH groups adjacent to the tetrahydrofuran (THF) rings resulted in a significant decrease in inhibitory potency. These structural dependencies of the inhibitory action of Deltalac-acetogenins are in marked contrast to those of ordinary acetogenins. Double-inhibitor titration of steady-state complex I activity showed that inhibition of compound 3 and bullatacin are not additive, though the inhibition site of both inhibitors is downstream of iron-sulfur cluster N2. Our results indicate that the mode of inhibitory action of Deltalac-acetogenins differs from that of ordinary acetogenins. Therefore, Deltalac-acetogenins can be regarded as a novel type of inhibitor acting on the terminal electron-transfer step of complex I.