Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase.

Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg(2+)-binding site, only Eß31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R ß subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R ß and DD ß subunits, Gluß97 of DD would coordinate to Mg(2+). The complex of Eß97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluß97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.

Effects of transforming growth factor-ß3 and matrix metalloproteinase-3 on the pathogenesis of chronic mitral valvular disease in dogs.

OBJECTIVE: To investigate the roles of transforming growth factor-ß (TGF-ß) isoforms and matrix metalloproteinases (MMPs) in development of chronic mitral valvular disease (CMVD) in dogs. SAMPLE POPULATION: 12 mitral valve leaflets collected from cadavers of 5 clinically normal dogs and from 7 dogs with CMVD. PROCEDURES: Expression of TGF-ß isoforms 1, 2, and 3; MMPs 1, 2, 3, and 9; TGF-ß receptor II (TßR-II); and α smooth muscle actin (αSMA) in mitral valves of dogs with CMVD was compared with that in mitral valves from clinically normal dogs. Additionally, responses of valvular interstitial cells (VICs) to TGF-ß3, MMP-3, and angiotensin-converting enzyme inhibitor (ACEI) as a suppressor of TGF-ß3 were examined in vitro. RESULTS: Expression of TGF-ß3, TßR-II, αSMA, and MMP-3 was only detected in mitral valves of dogs with CMVD. Concentrations of αSMA and proteoglycans in cultured VICs were significantly increased following incubation with TGF-ß3; treatment with MMP-3 resulted in increased amounts of active and total TGF-ß3, and total TGF-ß3 in VICs was significantly decreased by incubation with ACEI. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that increased TGF-ß3 and MMP-3 contribute to the pathogenesis of valvular degeneration associated with CMVD. In addition, it is possible that the use of ACEI could effectively block pathological alterations in VICs associated with CMVD in vitro. Impact on Human Medicine-CMVD is associated with primary mitral valve prolapse and Marfan syndrome in humans. Results of the study reported here will help to elucidate the molecular mechanisms of CMVD in dogs and humans.