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The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans , C. tropicalis , and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.

In an attempt to develop effective inhibitors of Candida secreted aspartic proteinases, we have prepared a series of N-protected peptides varying in the type of scissile bond replacement, in the P and P' side chains as well as in the N- and C-terminal modifications. The compounds were tested in vitro with the chromogenic peptide substrate using purified secreted proteinases of C. albicans and C. tropicalis. Our results have confirmed that the binding of inhibitors and their effectiveness is influenced by a number of enzyme-inhibitor interactions. Moreover, factors like solvation/desolvation contribute to the optimal binding energy of the inhibitors.

1. 1. The p15 gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (pr25 lac-Δgag ) was accompanied by its structurally correct processing. 2. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N -terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 4. 3. Contrary to reported data, the amino acid sequence of the p15 gag proteinase diners from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 5. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well at 2.3 A without deterioration.

The specificity of the p15 proteinase of myeloblastosis-associated virus (MAV) was tested with nonviral high molecular weight substrates and with synthetic peptides. Peptides with sequences spanning known cleavage sites in viral polyproteins of Rous sarcoma virus (RSV) and avian leukemia viruses, as well as in BSA and HSA, were synthesized, and the rate of their cleavage by the MAV proteinase was compared. Synthetic peptides require for successful cleavage at least 4 residues at the N-terminal side and 3 residues at the C-terminal side. The proteinase shows a preference for hydrophobic residues with bulky side chains (Met, Tyr, Phe) in P3, although Arg and Gln can also be accepted. Small hydrophobic residues are required in P2 and P2', and large hydrophobic residues (Tyr, Met, Phe/p-nitro-Phe) are preferred in both P1 and P1'. The difference between the specificity of the p15 proteinase and that of the HIV-1 proteinase mostly pertains to position P2' of the substrate, where bulkier side chains are accepted by the HIV-1 proteinase (Richards et al., 1990). A good chromogenic substrate for the MAV and RSV proteinases was developed and used to further characterize the MAV proteinase activity with respect to ionic strength and pH. The activity of the proteinase is strongly dependent on ionic strength and pH. Both the kcat and Km values contribute to a higher cleavage efficiency at higher salt concentrations and show a bell-shaped pH dependence curve with a sharp maximum at pH 5.5 (kcat) and 6.5 (Km).

Secreted aspartic proteases (Saps) of pathogenicCandidaspp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps fromC. albicans(Sap2p),C. tropicalis(Sapt1p), andC. parapsilosis(Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3′. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47–53 and 258–292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N-terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N- and C-termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10-10-10-9 M. The HIV-1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.

The key role of retrovirus-encoded proteinases in limited proteolysis of polyprotein precursors, a prerequisite of the formation of mature infectious virions, makes these enzymes attractive targets of specific inhibitors. A rational basis for the design of such inhibitors requires detailed knowledge of the proteinase-inhibitor interactions at the level of three-dimensional structures. Such data are available so far only for the HIV-1 proteinase whose crystal complexes with some inhibitors were subject of X-ray studies.1 The absence of such information on the myeloblastosis associated virus (MAV) proteinase, another retroviral aspartate proteinase, together with the need of finding a tight-binding yet hydrophilic inhibitor for both X-ray studies and titration of its active site, led us to investigate the interactions of the proteinase with various classes of custom-synthesized inhibitors.

All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases, are of similar size and of homologous primary structure; they all act catalytically in the form of highly symmetric molecular dimers.1 Detailed studies of retroviral proteinases were carried out on two almost identical proteinases of MAV2,3 and RS V4 (representing the group of avian retroviruses) and on the HIV proteinase.5,6 The knowledge of the 3D structure,2,4,5 catalytic activity and substrate specifity3,6 of the MAV and the HIV proteinase has changed the notion of their general similarity since several features that distinguish each proteinase from the other were revealed. The HIV-1 proteinase has a considerably higher activity3,6 which reflects the different conditions of the expression and action of this enzyme in vivo: 7 The “coding strategy” of MAV allows the expression of the proteinase from the first (gag) open reading frame and provides for the high (i.e. stoichiometrical) level of the relatively “weak” enzyme whereas the smaller amount of the more active HIV enzyme is a result of infrequent translational frameshift events that occur in the overlapping region of the gag and pol reading frames.8 The substrate specificities of retroviral proteinases seem complex and the requirement for a side chain in an individual subsite of a substrate is an outcome of the combination of residues occupying other closely located subsites.3 The two proteinases (MAV and HIV) show rather promiscuous substrate specificity, nevertheless several differences can be traced. We made an attempt to use protein engineering of the MAV proteinase to tackle directly problems of structural basis of these differences and, vice versa, to make more precise conclusions on the functional importance of the individual elements of its three dimensional structure. This article describes mutation of the MAV proteinase which resulted not only in an alteration of its substrate specificity but also in an increase of its enzymic activity — a rare case in protein engineering.

Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.

Candida is a typical opportunistic pathogen which is often present in healthy individuals but under certain conditions it causes fungal infections.1 In addition to superficial infections, Candida is a major cause of deep systemic infections in immunocompromised patients such as HIV patients, transplant recipients, or cancer patients undergoing chemotherapy. An aspartic proteinase secreted by many members of the genus Candida has been often suggested to take part in the invasive character of the microorganism.2 The aspartic proteinases secreted by Candida represent a potential target for the drug intervention of the disease and the studies devoted to the understanding of these enzymes span from genetic studies3 to substrate specificity studies4 or crystallographic studies.5,6 It has been shown that there are differences in the specificity of secreted aspartic proteinases (SAP) from different Candida strains and that these proteinases are in general similar to eukaryotic aspartic proteinases with a deep active site cleft which accommodates at least eight residues of a substrate or inhibitor.