Specific cellular proteins associate with angiotensin-converting enzyme and regulate its intracellular transport and cleavage-secretion.

Angiotensin-converting enzyme (ACE) is an extensively glycosylated type I ectoprotein anchored in the plasma membrane by a hydrophobic transmembrane domain. In tissue culture as well as in vivo, the extracellular domain of ACE is released into the culture medium by a regulated proteolytic cleavage. To identify the cellular proteins that regulate ACE processing and cleavage-secretion, ACE-bound proteins were purified by affinity chromatography and characterized by microsequencing and Western blotting. One protein was identified as ribophorin and another as immunoglobulin-binding protein (BiP), a chaperone. Metabolic labeling and immunoprecipitation of ACE confirmed its interaction with BiP. Overexpression of BiP inhibited ACE secretion, an effect accentuated by the expression of an enzymatically inactive mutant BiP. This inhibition was caused by the retention of ACE precursors by BiP in the endoplasmic reticulum, as revealed by immunoprecipitation and immunofluorescence experiments. However, treatment with a phorbol ester, phorbol 12-myristate 13-acetate, enhanced ACE secretion even from cells overexpressing BiP. Western blot analysis of ACE-associated proteins with antibodies to protein kinase C (PKC) revealed the presence of its specific isozymes. Treatment with phorbol 12-myristate 13-acetate caused marked reduction in ACE association of selective PKC species. Thus, our studies have identified PKC and BiP as two proteins that directly interact with ACE and modulate its cell-surface expression and cleavage-secretion.

Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice.

Mammalian angiotensin-converting enzyme (ACE) is one of several biologically important ectoproteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic cleavage. It has been suggested that a common proteolytic system is responsible for the cleavage of a diverse group of membrane ectoproteins, and tumor necrosis factor-alpha-converting enzyme (TACE), a recently purified disintegrin-metalloprotease, has been implicated in the proteolytic cleavage of several cell surface proteins. Mice devoid of TACE have been developed by gene targeting. Such mice could provide a useful system to determine if TACE is responsible for the cleavage of other ectoproteins. Cultured fibroblasts without TACE activity, when transfected with cDNA encoding for the testicular isozyme of ACE (ACET), synthesized and secreted ACET normally after a proteolytic cleavage near the C terminus. In addition, similar quantities of the soluble, C-terminally truncated somatic isozyme of ACE (ACEP) were present in the serum of wild-type and TACE-deficient mice. These results demonstrate that TACE is not essential in the generation of soluble ACE under physiological conditions. Finally, we also report solubilization of ACE-secretase, the enzyme that cleaves ACE, from mouse ACE89 cells and from rabbit lung. We demonstrate that soluble ACE-secretase from both sources failed to cleave its substrate in solution, suggesting a requirement for anchoring to the membrane.

Characterization of the respiratory chain of Leishmania donovani promastigotes.

Inhibition analysis of respiration of Leishmania donovani promastigotes in resting, starved and permeabilized cells in the presence of classical electron transfer complex inhibitors such as rotenone, thenoyltrifluoroacetone and antimycin demonstrated the absence of complex I component of the respiratory chain in this organism. Cyanide failed to completely block the oxygen uptake (residual 25-30%) even at high concentrations. The alternative oxidase inhibitor for Trypanosoma brucei, salicylhydroxamic acid (SHAM) had no effect on respiration while the cytochrome o inhibitor orthohydroxydiphenyl (OHD) could block cyanide-insensitive respiration at low concentrations. Succinate-dependent O2 uptake in permeabilized cells follows the classical pathway. Oxidation of NADH by a membrane-rich fraction produced H2O2 as the end product and was insensitive to respiratory chain inhibitors. The presence of NADH-fumarate reductase was demonstrated in membrane-rich fraction and fumarate could reduce H2O2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrons from NADH. A differential route for NADH oxidation was further confirmed by NADH cytochrome c reductase insensitivity to antimycin. A tentative scheme for electron transfer pathway in this organism is proposed in which a reversal of Krebs cycle enzymes occur producing succinate that can be excreted or oxidized depending upon the energy demands of the cell. Inhibition studies also suggest bifurcations of the respiratory chain that can be of minor importance for the organism.

Biosynthesis of isoprenoid compounds in cattle filarial parasite Setaria digitata.

The biological significance of isoprenoid compounds such as ubiquinones, prenols and sterols have been well established. The presence and biological function of the two quinones Q6 and Q8 in the cattle filarial parasite Setaria digitata have already been reported. Inhibition of the function of quinone was already shown to be an effective means of controlling the filarial parasite. Detailed investigations of the non-saponifiable lipids from S. digitata using column, thin layer, reverse phase and high performance liquid chromatography showed the presence and formation of isoprenoid compounds such as prenols and sterols, in addition to the two quinones. Blocking of the biosynthesis of these useful compounds may prove to be an additional means of control of filarial parasites.

Quinone analogues: a drug of choice for the control of filariasis.

Human filariasis is caused by Wuchereria bancrofti, Brugia malayi and B. timori. Of the several recommended model filarial parasites by WHO, Setaria digitata a bovine one has characteristics such as cyanide insensitivity, lack of detectable cytochromes, presence of two quinones Q8 and Q6. Of the two quinones Q8 seems to have a predominant role in energy production. In vitro inhibitory studies using quinone analogues, coenzyme Q0 and menadione have shown that these compounds paralyse the worms in very low concentrations compared to diethyl carbamazine, the drug of choice for filariasis. The mitochondrial energy production associated with electron transfer is intercepted by quinone analogues. Hence for the treatment of filariasis, this study paves a chemotherapeutic target for the design of drugs which can control the parasites by interacting at the subcellular level by energy depletion.

Oxidative phosphorylation coupled to electron transfer in the filarial parasite Setaria digitata.

The filarial parasite of Bos indicus, Setaria digitata is reported to have many unique characteristics such as cyanide insensitivity and mitochondrial hydrogen peroxide production. The latter is more sensitive to the alternative oxidase inhibitor salicylhydroxamic acid (SHAM). Studies on the generation of ATP molecules through mitochondriae in the presence of different substrates and inhibitors showed that the oxidative phosphorylation coupled to electron transport occurs mainly at site I and involves the participation of quinone Q8. Based on the data, a scheme for the filarial electron transport system is proposed in which the quinones have a central role. Hence inhibitors at the quinone level might prove to be effective targets for chemotherapy.

Quinone mediated electron transport system in the filarial parasite Setaria digitata.

Setaria digitata, a cattle filarial parasite, is known to have peculiarities such as hydrogen peroxide (H2O2) production, cyanide insensitivity, absence of cytochromes and presence of quinones. Estimation of mitochondrial H2O2 with different substrates and inhibitors showed that salicylhydroxamic acid (SHAM), the alternative oxidase inhibitor, inhibited the H2O2 production maximally. Based on the inhibitory studies with rotenone, antimycin A, o-hydroxydiphenyl, SHAM and 2 thenoyltrifluoroacetone, a mechanism for the electron transport is proposed. Quinone Q8 seems to have a central role, hence inhibitors at the level of quinones might prove to be effective in designing drugs for filariasis.

Quinone mediated ATP production in the filarial parasite Setaria digitata.

The cattle filarial parasite Setaria digitata, a facultative anaerobe which is reported to be cyanide insensitive, lacks cytochromes and presents many unique characters. Experiments showed the occurrence of two lower quinones Q6 and Q8 and its rapid synthesis is revealed by a [14C] acetate incorporation study. A schematic quinone mediated hydrogen peroxide production with the generation of ATP through oxidation of substrates has been proposed. Search for specific blockers at the level of quinone might prove to be an effective measure for the control of filarial parasites and thereby filariasis.