The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop.     

Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis

Efficient clearance of mucus and inhaled pathogens from the lung is dependent on an optimal airway surface liquid (ASL) volume, which is maintained by the regulated transport of sodium and chloride across the airway epithelium. Accumulating evidence suggests that impaired mucus clearance in cystic fibrosis (CF) airways is a result of ASL depletion caused by excessive Na(+) absorption through the epithelial sodium channel (ENaC). However, the cellular mechanisms that result in increased ENaC activity in CF airways are not completely understood. Recently, proteases were shown to modulate the activity of ENaC, but the relevance of this mechanism to the physiologic regulation of ASL volume is unknown. Using primary human airway epithelial cells, we demonstrate that: (i) protease inhibitors are present in the ASL and prevent the activation of near-silent ENaC, (ii) when the ASL volume is increased, endogenous protease inhibitors become diluted, allowing for proteolytic activation of near-silent channels, and (iii) in CF, the normally present near-silent pool of ENaC is constitutively active and the alpha subunit undergoes increased proteolytic processing. These findings indicate that the ASL volume modulates the activity of ENaC by modification of the serine protease-protease inhibitor balance and that alterations in this balance contribute to excessive Na(+) absorption in cystic fibrosis.     

Profilin inhibits pollen tube growth through actin-binding,but not poly-<Emphasis Type="SmallCaps">l</Emphasis>-proline-binding

Previously, we have shown that excess profilin inhibits pollen tube growth at significantly lower concentrations than it blocks cytoplasmic streaming. To elucidate the mechanism by which profilin achieves this function, we have employed mutant profilins from Schizosaccharomyces pombe [J. Lu and T.D. Pollard (2001) Mol Biol Cell 12:1161–1175], which have defects in actin-binding, ability to inhibit polymerization, and poly-l-proline (PLP)-binding. Using Lilium longiflorum L. pollen and S. pombe profilins as wild-type (wt) standards, mutant profilins have been injected into pollen tubes of Lilium, and examined for their effects on growth rate and cell morphology. Our results show that mutant Y5D (68% actin-binding; 1.1% PLP-binding) is indistinguishable from wt-standard profilins. However mutant K81F (2.7% actin-binding; 77% PLP-binding) and especially mutant K67E (<1% actin-binding; 100% PLP-binding) are significantly less effective than wt-standard profilins in their ability to inhibit pollen tube growth. PLP also inhibits pollen tube growth. However, PLP is not different from K67E/PLP combined, which has no actin-binding, suggesting that PLP does not function by binding to profilin. In addition, there are differences in the morphology and F-actin organization in cells injected with PLP versus wt-profilin. Whereas wt-profilin causes a fragmentation and marked reduction in the amount of F-actin [L. Vidali et al. (2001) Mol Biol Cell 12:2534–2545], PLP generates an extensive disorganization without any apparent reduction in the amount of F-actin. We conclude that along with actin-binding activity of profilin, PLP-containing proteins also participate in the growth control process, and can do so independently of binding to profilin.Abbreviations 3D Three-dimensional - PLP Poly-l-proline - RMS Root mean square - wt Wild type     

Analysis of the functional role of a G.A sheared base pair by in vitro genetics

A classical genetic strategy has been combined with an in vitro selection method to search for functional interactions between the two domains of the hairpin ribozyme. G(21) is located within internal loop B; it is proposed to form a sheared base pair with A(43) across loop B and to bind a Mg(2+) ion. Both nucleotides are important for ribozyme function, and G.A sheared base pairs are a very widespread motif in structured RNA. We took advantage of its presence in the hairpin ribozyme to study its functional role. Pseudorevertants, in which the loss of G(21) was compensated by mutations at other positions, were isolated by in vitro selection. The vast majority of G(21) revertants contained substitutions within domain A, pointing to functional communication between specific sites within the two domains of the hairpin ribozyme. The possibility of a direct or redundant contacts is supported by electrophoretic mobility shift studies showing that a complex formed between domain B of the ribozyme and the substrate was disrupted and restored by base substitutions that have analogous effects on catalytic activity. The functional significance of this complex, the role of the nucleotides involved, and the basis for magnesium ion requirement is discussed.     

Localisation and functional studies on the 5′ -nucleotidase of the cattle tick Boophilus microplus

The ecto-5-nucleotidase from the cattle tick Boophilus microplus is an unusual enzyme, hydrolysing a variety of nucleoside mono-, di- and triphosphates to release the free nucleoside. The gene has been sequenced and the recombinant protein expressed as a functional, active enzyme. Nevertheless, the function of the enzyme in the tick remains obscure. The enzyme is present throughout the life cycle, but in largest amounts in unfed larvae and adult ticks. The tissue location has been studied in adult female ticks by Western blotting, RT-PCR and immunofluorescence. All methods show the enzyme to be principally in the Malpighian tubules, though significant amounts are also present on the surface of ovaries and in detectible amounts in other tissues. This, together with the known specificity of the enzyme, suggests a role in purine salvage pathways. Sensitivity of ticks to allopurinol, an inhibitor of hypoxanthine-guanine-phosphoribosyltransferase, supports the importance of purine salvage in this tick and the potential role of nucleotidase in this pathway.