Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.
Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid. Examination of strains expressing L27 variants truncated at the N terminus reveals that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in peptidyl transferase activity, and a sharp decline in the labeling of L27 from the 3' end of a photoreactive tRNA at the ribosomal P site. These findings suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the peptidyl transferase active site and contribute to its function, possibly by helping to correctly position tRNA substrates at the catalytic site.
SummaryIn contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY III , cheA II , cheW II , cheW III , cheR II , cheB and tlpC. When introduced into a wild-type background, deletion of cheA II produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA II forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that downregulates low-density lipoprotein (LDL) receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C). Phenotypic screening of a small-molecule compound collection was used to identify an inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), which was shown to stimulate uptake of LDL-C in hepatoma cells by increasing LDL-R levels, without altering levels of secreted transferrin. Systematic investigation of the mode of action revealed that R-IMPP did not decrease PCSK9 transcription or increase PCSK9 degradation, but instead caused transcript-dependent inhibition of PCSK9 translation. In support of this surprising mechanism of action, we found that R-IMPP was able to selectively bind to human, but not E. coli, ribosomes. This study opens a new avenue for the development of drugs that modulate the activity of target proteins by mechanisms involving inhibition of eukaryotic translation.
SUMMARY The assembly of bacterial ribosomes is viewed with increasing interest as a potential target for new antibiotics. The in vivo synthesis and assembly of ribosomes are briefly reviewed here, highlighting the many ways in which assembly can be perturbed. The process is compared with the model in vitro process from which much of our knowledge is derived. The coordinate synthesis of the ribosomal components is essential for their ordered and efficient assembly; antibiotics interfere with this coordination and therefore affect assembly. It has also been claimed that the binding of antibiotics to nascent ribosomes prevents their assembly. These two contrasting models of antibiotic action are compared and evaluated. Finally, the suitability and tractability of assembly as a drug target are assessed.
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