Structure–activity relationship studies of Niemann-Pick type C1-like 1 (NPC1L1) ligands identified by screening assay monitoring pharmacological chaperone effect

A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure–activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.     

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.     

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser⁶⁵ by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.     

Heterologous expression of tomato glycoside hydrolase family 3 α‐L‐arabinofuranosidase/β‐xylosidases in tobacco suspension cultured cells and synergic action of a family 51 isozyme under antisense suppression of the enzyme

Four cDNA clones (SlArf/Xyl1‐4) encoding α‐l ‐arabinofuranosidase/β‐xylosidase belonging to glycoside hydrolase family 3 were obtained from tomato (Solanum lycopersicum) fruit. SlArf/Xyl1 was expressed in various organs. Its level was particularly high in flower and leaves but low in fruit. SlArf/Xyl3 was highly expressed in flower. On the contrary, SlArf/Xyl2 and 4 were expressed in early developmental stage in various organs. Comparison with SlArf/Xyl4, SlArf/Xyl2 expression was observed in earlier stages. The active recombinant proteins were obtained by using BY‐2 tobacco (Nicotiana tabacum) suspension cultured cells. The SlArf/Xyl1 and 2 recombinant proteins showed a bi‐functional activity of α‐l ‐arabinofuranosidase/β‐xylosidase while the SlArf/Xyl4 protein possessed a β‐xylosidase activity predominantly. Neither enzyme activities were detected for the SlArf/Xyl3 protein under the same conditions. Although SlArf/Xyl2 possessed a bi‐functional activity, it preferentially hydrolyzed arabinosyl residues from tomato hemicellulosic polysaccharides. Antisense suppression of SlArf/Xyl2 resulted in no apparent changes in the enzyme activities, monosaccharide composition or fruit phenotype. Increment of a family 51 α‐l ‐arabinofuranosidase expression rather than that of family 3 resulted in a restoring the activity in SlArf/Xyl2‐suppressed fruit. The ability of recombinant SlArf/Xyl2 to hydrolyze both arabinan and arabinoxylan is nearly identical to that of α‐l ‐arabinofuranosidases belonging to family 51. Our results suggested that BY‐2 cells are a useful expression system for obtaining active cell wall hydrolyzing enzymes. In addition, an α‐l ‐arabinofuranosidase activity derived from SlArf/Xyl2 would be essential in young organ development and the action of the enzyme could be restored by the other enzyme belonging to a different family under a defective condition.     

Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A–H2B and DNA association with the G. lamblia H3–H4 were weaker than those for human H3–H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.     

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration

Using N₂ cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate–ubiquinone reductase/quinol–fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol–fumarate reductase.     

Effects of salmon calcitonin and anti-salmon calcitonin antibody on plasma calcium concentrations in the goldfish,Carassius auratus

The role of calcitonin (CT) in plasma calcium regulation was studied by the administration of exogenous CT and anti-salmon(s) CT antibody using goldfish,Carasius auratus, loaded or otherwise with calcium. CT elicited a decrease in plasma calcium concentrations at a dose of 10 ng/g body weight 1 h after administration. However, no effects were observed following doses of 30 ng and 50 ng/g 1 h, nor for the three doses 3 h after administration. In calcium-loaded fish, the effect of CT was different depending on the dosage of CT. Ten ng and 50 ng/g induced a decrease and an increase in plasma calcium concentrations, respectively, 3 h after administration. Anti-sCT antibody (0.02 μg or 0.1 μg/g) did not affect plasma calcium concentrations. In calcium-loaded fish, neither dose of anti-sCT antibody changed plasma calcium concentrations 1 h after administration. However, following a dose of 0.1 μg/g, plasma calcium concentrations decreased after 3 h. A positive correlation between plasma calcium concentrations and the gonad somatic index (GSI) in females was no longer apparent after administration of anti-sCT antibody. There was no relationship between plasma calcium concentrations and GSI in control and anti-sCT antibody-treated males. These results suggested that CT regulates plasma calcium concentrations in different ways depending on the dosage with CT having a role in calcium physiology during vitellogenesis.