Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.     

Combined Analysis of Variation in Core,Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug–resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.     

Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σ^I) factors that have cognate membrane-associated anti-σ^I factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σ^I4-dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σ^I3-dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14^A) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.     

Early effects of serum on phospholipid metabolism in untransformed and oncogenic virus-transformed cultured fibroblasts.

The addition of serum to previously serum-deprived 3T3 fibroblasts in culture caused a pronounced, rapid and selective stimulation of the incorporation of [³²P]phosphate into phosphatidyl inositol. Comparison of the content of radioactivity in phosphatidyl inositol after a short pulse with that obtained following a prolonged labeling period showed that serum accelerated the rate of the turnover (and not the net accumulation) of this substance. In cells transformed by SV-40 virus, the rate of labeling of phosphatidyl inositol was relatively high, and was not influenced significantly by the deprivation of serum or its resupplementation. It is suggested that the rate of phosphatidyl inositol turnover may be related to the state of the mobility of membrane constituents, and that this process escapes the control of serum factors in malignantly transformed cells.     

Naloxone effects on a nociceptive response of hypophysectomized and adrenalectomized mice

Naloxone (1 and 3 mg/kg) or saline was administered to adrenalectomized, sham-adrenalectomized, hypophysectomized, and sham-hypophysectomized mice prior to placing them on a 55° C hot plate. Naloxone reduced the jump latency of the adrenalectomized, sham-adrenalectomized, and sham-hypophysectomized mice, but did not reduce the jump latency of hypophysectomized mice. Hypophysectomy and adrenalectomy $\text{per se}$ did not significantly affect the jump latency. These results indicate that the pituitary is necessary for naloxone to reduce the escape latency of mice on the hot plate. The possibility of a hyperalgesic factor is suggested.     

Imprinted methylation and its effect on expression of the mouse Zfp127 gene.

The Zfp127 gene is located on mouse chromosome 7 in an imprinted region that is homologous to the 2-Mb Prader-Willi and Angelman Syndromes region on human chromosome 15q11-q13. Here, we show that the gene is differentially methylated, the maternal allele being methylated and the paternal allele being unmethylated. This maternal methylation is established promptly after fertilization prior to syngamy. We also provide data that demonstrate the significance of methylation in the paternal expression of the gene. The expression of the Zfp127 gene in methyltransferase-deficient mice is significantly higher, suggesting that the gene is biallelically expressed in these mice. The data presented here will help to understand the mechanism by which the monoallelic expression of the entire 2-Mb Prader-Willi and Angelman Syndrome region is regulated.     

A yeast strain that uses D-galacturonic acid as a substrate for L-ascorbic acid biosynthesis

Summary The bioconversion of D-galacturonic acid to L-ascorbic acid was demonstrated in a new yeast strain isolated from the Japanese Crystal. Both intact cells and a crude mitochondrial extract yielded L-ascorbic acid when D-galacturonic acid was present.     

The pleiotypic response in mammalian cells: search for an intracellular mediator

The reactions included in the definition of this regulatory program, called the “pleiotypic response”, are very similar to those under “stringent” control in bacteria. The latter appears to be mediated by a novel nucleotide, ppGpp, and we therefore assayed for the presence of this nucleotide in cultured fibroblasts under a variety of conditions of growth restriction which elicit the pleiotypic response. We were unable to detect it in any of these circumstances and we conclude that ppGpp is not the mediator of the pleiotypic response, at least in the cell lines studied.