Schematic representation of the influence of magnesium and potassium on the degradation of DNA in CF mucus by rhDNase I. CF mucus contains actin, which can be in the globular (G-actin, Mr 42,000) or in the polymer, i.e., filamentous (F) form. G-actin is a potent inhibitor (Kä~1 nM) of DNase I and magnesium is known to promote the polymerization of G-actin into F-actin, which does not inhibit DNase I.

inhibitor of DNase I [112] (Figure 5.7). Although the amount of G-actin in CF mucus is not well defined, filamentous actin (F-actin), which is in equilibrium with G-actin, is present in CF mucus at concentrations ranging from 0.06 to 5 mg/mL [52,111].

On the basis of ternary model of the DNase I-actin-DNA complex, Ulmer, et al. [113] showed that actin binds DNase I near the DNA-binding site [113]. Consequently, the inhibition of DNase I by actin is most likely due to a steric blockage of the active site of DNase I [113,114]. Binding of G-actin to DNase I involves hydrogen bonds and electrostatic interactions between E13, H44, D53, Y65, V67, E69, A114 of DNase I and T203, E207/G63, R39, Q41/V43, V45, K61, V45 of G-actin [113,115,116]. Additionally, the actin residues G42, V43, and M44 are incorporated as a parallel strand of a ß-pleated sheet in DNase I with partners Y65, V66, and V67. The DNase I-actin complex (binding constant 5 x 108 M-1) can be separated by 5'-nucleotidase, resulting in a reactivation of DNase I [117,118]. In CF mucus, the inhibition of rhDNase I by actin has also been overcome by actin-binding proteins (gelsolin), and by actin-resistant variants of human DNase I [113,119]. The latter were created by substitution of the amino acids of rhDNase I involved in actin binding by charged, oppositely charged, or bulky amino acids [113]. These actin-resistant variants are 10- to 50-fold more potent in CF mucus than in wild-type rhDNase I [120]. Additionally, different naturally occurring actin-resistant forms of human DNases I and II, such as LSDNase, and DNase II-like acid DNase (DLAD), have been discovered and proposed as suitable candidates to substitute the actin-sensitive rhDNase I in the treatment of CF [121-123]. Moreover, the finding that DLAD is a secreted enzyme that is also expressed in the human lung led to the speculation that DLAD is responsible for normal DNA clearance in the airways and that the CF defect may result in reduced secretion or activity of the endogenous DLAD [123].

Was this article helpful?

0 0

Post a comment