Aspartic Peptidases And Disease

Aspartic proteinases may play many roles in human disease, although in many cases the speculations outnumber the proofs. For many years, gastric pepsin was suspected of contributing to the formation of stomach ulcers. In cases of cancer of the esophagus, pepsinogen secretion is stimulated and the excessive amounts of pepsin that result could play a role in extensive degradation of the esophagus and upper stomach (Varis et al., 1991).

Human cathepsin D has long been suspected of involvement in breast cancer, again due to its abundant secretion in necrotic tissues (Rochefort et al., 2000). However, it is unclear whether this is a cause or a consequence of the lesions. Furthermore, the activity in an extracellular environment that is not acidic could be limiting. The enzyme is over-expressed in some tumor cell lines.

Renin plays a role in regulation of blood pressure in mammals, and has long been the target of drug discovery for control of hypertension. In fact, the rapid advances in the discovery of effective drugs for HIV infection, with HIV protease as the target, were largely based on prior experience in studies of renin.

The mamepsins, recently reported membrane-bound enzymes, have been implicated in the aberrant processing of beta amyloid protein to yield the fragments that precipitate and contribute to plaque formation that leads to Alzheimer's Disease.

Microorganisms that cause human disease are numerous and many employ aspartic pro-teinases to gain entry into cells and tissues. Fungi such as Candida albicans produce secreted enzymes of this class that have been shown to be virulence factors for disease. The chestnut blight fungus, Cryphonectria parasitica, produces endothiapepsin, whose structure was one of the first members of the family to be solved by X-ray crystallography (Blundell et al., 1990). The fungus has a devastating effect upon the American chestnut tree.

As mentioned earlier, aspartic peptidases in the malaria parasite, Plasmodium falciparum, may be involved in the supply of nutrients derived from hemoglobin degradation. Cysteine proteinases are also involved in this process, and the recent discovery of several additional genes has complicated the scenario.

RECOMBINANT EXPRESSION, PURIFICATION, AND ASSAYS OF ASPARTIC PEPTIDASES

In addition to purification of naturally occurring enzymes from extracellular secretions or tissues, aspartic proteinases can be produced by recombinant means. Expression in bacteria, plant cells, yeast, and mammalian systems has been reported for various members of the family. In general, expression of the proenzyme form is necessary in order to permit proper folding of the polypeptide chain. Several early attempts to produce the mature form of the enzyme, lacking the 40- to 50-residue prosegment, were unsuccessful, whereas the proenzymes can be produced in inclusion bodies in bacterial expression systems and refolded. The yields are highly variable, depending on the sequence, and there is a need for improvements in the expression of many enzymes of this family.

Assays of enzymes in this class vary from the classical Anson hemoglobin digestion assay, where the absorbance of solubilized peptide fragments is quantified, to high throughput fluorescence assays utilizing synthetic peptides. General and specific assays for pepti-dases will be described in other units in this chapter.

Literature Cited

Barrett, A.J., Rawlings, N.D., and Woessner, J.F. 1998. The Handbook of Proteolytic Enzymes, pp. 799-986. Academic Press, San Diego.

Blundell, T.L., Jenkins, J.A., Sewell, B.T., Pearl, L.H., Cooper, J.B., Tickle, I.J., Veerapandian, B., and Wood, S.P. 1990. X-ray analysis of aspartic proteinases: The 3-dimensional structure at 2.1 A resolution of endothiapepsin. J. Mol. Biol. 211:919-941.

Davies, D.R. 1990. The structure and function of the aspartic proteinases. Annu. Rev. Biophys. Chem. 19:189-215.

Francis, S.E., Sullivan, D.J., and Goldberg, D.E. 1997. Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. Annu. Rev. Microbiol. 51:97-123.

Fraser, M.E., Strynadka, N.C.J., Bartlett, P.A., Hanson, J.E., and James, M.N.G. 1992. Crystal-lographic analysis of transition-state minics bound to penicillopepsin-phosphorus-contain-ing peptide analogs. Biochemistry 31:52015214.

Green, D.W., Aykent, S., Gierse, J.K., and Zupec, M.E. 1990. Substrate specificity of recombinant human renal renin: Effect of histidine in the P2 subsite on pH dependence. Biochemistry 29:3126-3133.

Peptidases

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