Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

DDX23, an Evolutionary Conserved dsRNA Sensor, Participates in Innate Antiviral Responses by Pairing With TRIF or MAVS.

DExD/H-box helicases play essential roles in RNA metabolism, and emerging data suggests that they have additional functions in antiviral immunity across species. However, little is known about this evolutionarily conserved family in antiviral responses in lower species. Here, through isolation of poly(I:C)-binding proteins in amphioxus, an extant basal chordate, we found that DExD/H-box helicases DHX9, DHX15, and DDX23 are responsible for cytoplasmic dsRNA detection in amphioxus. Since the antiviral roles of DDX23 have not been characterized in mammals, we performed further poly(I:C) pull-down assays and found that human DDX23 binds to LMW poly(I:C) through its N-terminal region, suggesting that DDX23 is an evolutionarily conserved dsRNA sensor. Knockdown of human DDX23 enhanced the replication of VSV and reduced the activation of the NF-κB and IRF3. Moreover, when stimulated with poly(I:C) or VSV, human DDX23 translocated from the nucleus to the cytoplasm and formed complexes with TRIF or MAVS to initiate downstream signaling. Collectively, this comparative immunological study not only defined DDX23 as an emerging nuclear pattern recognition receptor (PRR) for the innate sensing of an RNA virus, but also extended the essential role of the DExD/H helicase family in viral RNA sensing from mammals to basal chordates.

hsa-miR-96 up-regulates MAP4K1 and IRS1 and may function as a promising diagnostic marker in human bladder urothelial carcinomas.

Numerous microRNAs (miRNAs) play crucial roles in cancer development. In this study, we report that hsa-miR-96 is expressed at higher levels in human bladder urothelial carcinomas compared to normal tissues. We found that hsa-miR-96 increased invasion and differentiation of human bladder T24 cells and promoted their growth. Down­regulation of hsa-miR-96 significantly affected the phenotype of bladder cancer T24 cells. The mRNA and protein levels of insulin receptor substrate 1 (IRS1) and MAP4K1 were significantly reduced in cells transfected with the hsa-miR-96 inhibitor when compared with levels in cells transfected with the empty plasmid vector or the negative control miRNA inhibitor. Altogether, these results suggest that hsa-miR-96 may affect the growth of bladder cancer cells by up-regulating IRS1 and MAP4K1 levels, functioning as a promising diagnostic marker in human bladder urothelial carcinomas.

[Phloroglucinol: safe and effective for the prevention of bladder spasm after TURP].

OBJECTIVE: To evaluate the efficacy of phloroglucinol in preventing bladder spasm after transurethral resection of the prostate (TURP). METHODS: Using the random sampling method, we assigned 74 cases of TURP into a treatment group (n = 39), given 80 mg phloroglucinol every day for 3 days, and a control group (n = 35), left untreated. Then we observed the frequency, duration and pain of bladder spasm within the 3 days and compared them between the two groups. RESULTS: The mean frequency, duration and pain visual analogue score of bladder spasm were (4.3 +/- 1.2) times, (7.2 +/- 2.1) min and 3.2 +/- 1.6 respectively in the treatment group, as compared with (7.5 +/- 2.4) times, (15.6 +/- 6.8) min and 4.7 +/- 2.3 in the control, with statistically significant differences between the two groups (P < 0.05). And no obvious adverse reactions were found in the treatment group. CONCLUSION: Phloroglucinol is safe and effective for the prevention and treatment of bladder spasm following TURP.