ChangWoo Lee

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor.

The angiotensin II type I (AT1) receptor mediates regulation of blood pressure and water‐electrolyte balance by Ang II. Substitution of Gly for Asn111 of the AT1 receptor constitutively activates the receptor leading to Gq‐coupled IP3 production independent of Ang II binding. The Ang II‐activated conformation of the AT1N111G receptor was proposed to be similar to that of the wild‐type AT1 receptor, although, various aspects of the Ang II‐induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild‐type and the constitutively active AT1 receptors are different. Upon Ang II binding an activated conformation of the wild‐type AT1 receptor activates G protein and recruits β‐arrestin. In contrast, the agonist‐bound AT1N111G mutant receptor preferentially couples to Gq and is inadequate in β‐arrestin recruitment.

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

Field Bioassay for Longhorn Pine Sawyer Beetle Monochamus alternatus (Coleoptera: Cerambycidae) in Korea Based on Aggregation Pheromone 2-(Undecyloxy)ethanol

The pinewood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) poses a serious threat to pine forests in Europe and East Asia, leading to a debilitating pine wilt disease. Infected pine trees in Korea are generally fumigated or crushed to small wood chips after felling. Although pine wilt disease often recurs in pest management sites, there are no adequate means to monitor the effectiveness of pest control measures in those sites. Recently, a male-produced aggregation pheromone, 2-(undecyloxy)ethanol, was shown to be useful for attracting several Monochamus species, which are vectors for the pinewood nematodes. In this study, we investigated the abilities of 2-(undecyloxy)ethanol at three different doses (175, 350, and 700 mg), as well as host plant volatiles (α-pinene and ethanol), to attract M. alternatus (Coleoptera: Cerambycidae) at a pine forest in Pohang, Korea where infected pine trees had been cut down and fumigated. Twenty-seven M. alternatus were captured in cross-vane panel traps made of polyethylene terephthalate bottles and acrylic sheets. The results indicate that a high dose of 2-(undecyloxy)ethanol (700 mg per trap) is the most effective for attracting M. alternatus. The aggregation pheromone could be used to monitor the effectiveness of pest control measures as well as M. alternatus populations.

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin.

Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for GD(4)K-AMC, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of GD(4)K-AMC hydrolysis was pH 8.0. Our data indicate that GD(4)K-AMC is more suitable as a substrate for enteropeptidase than GD(4)K-NA.

Identification of Phosphoproteins Induced by AT1 Receptor Blocker Losartan

The angiotensin Ⅱ receptor (AT₁R) antagonists are effective in treating patients with hypertension and showed beneficial effects in diabetes and other metabolic diseases. The beneficial effects of AT₁R antagonists are mainly considered to be from inhibition of Ang Ⅱ-AT₁R signaling pathway such as the activation of NADPH oxidase and the generation of reactive oxygen species. In this study, we examined whether antagonist of the AT1R could account for phosphorylation of proteins in cells using antibody array. We have selected 6 proteins with Ser/Thr-phosphorylation sites and 12 proteins with Tyr-phosphorylation sites based on literature search. Upon AT1R antagonist losartan treatment to serum-starved COS-1 cells, there was ～20% increase of Ser phosphorylation in small GTPase RhoA. RhoA is known to be responsible for cytoskeleton rearrangement and is down-regulated upon Ser phosphorylation in vivo. Our finding provides a new insight into the mechanism and signaling pathway of the AT1R antagonist in cells.