Fungal gene-encoded peptidase inhibitors.

Peptidases can be inhibited by natural or synthetic small-molecule compounds, or by gene-encoded, proteinaceous inhibitors. Small-molecule peptidase inhibitors have been in the spotlight of researchers and pharmaceutical companies for many years. The studies concerning gene-encoded inhibitors are less frequent. The last decade has seen a boom of fungal genomics followed by extensive bioinformatic analyses focused particularly on those species that can cause infections in humans, animals or crops. Many sequences of putative inhibitors have been identified on the basis of homology with gene-encoded peptidase inhibitors of Saccharomyces cerevisiae, mammals or other organisms. However, characterization of the respective proteins is often missing. Gene-encoding peptidase inhibitors are rather diverse in size, mode of action, type of the target peptidase and localization. While some of the inhibitors are secreted to extracellular space and participate in host-pathogen interactions, others act intracellularly and their precise role in fungal physiology is not fully understood. However, most of the gene-encoded peptidase inhibitors are rather selective and efficient, and may be an inspiration for future directions of antimycotic research.

Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors.

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.

Cleavage of vimentin by different retroviral proteases.

Proteases (PRs) of retroviruses cleave viral polyproteins into their mature structural proteins and replication enzymes. Besides this essential role in the replication cycle of retroviruses, PRs also cleave a variety of host cell proteins. We have analyzed the in vitro cleavage of mouse vimentin by proteases of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (M-PMV), myeloblastosis-associated virus (MAV), and two active-site mutants of MAV PR. Retroviral proteases display significant differences in specificity requirements. Here, we show a comparison of substrate specificities of several retroviral proteases on vimentin as a substrate. Vimentin was cleaved by all the proteases at different sites and with different rates. The results show that the physiologically important cellular protein vimentin can be degraded by different retroviral proteases.

Three active forms of aspartic proteinase from Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus (M-PMV) proteinase, released by the autocatalytic cleavage of Gag-Pro and Gag-Pro-Pol polypeptide precursors, catalyzes the processing of viral precursors to yield the structural proteins and enzymes of the virion. In retroviruses, usually only one proteolytically active form of proteinase exists. Here, we describe an unusual feature of M-PMV, the existence of three active forms of a retroviral proteinase with molecular masses of 17, 13, and 12 kDa as determined by mass spectroscopy. These forms arise in vitro by self-processing of a 26-kDa proteinase precursor. We have developed a process for isolation of each truncated product and demonstrate that all three forms display proteolytic activity. Amino acid analyses, as well as the determination of N- and C-terminal sequences, revealed that the N-termini of all three forms are identical, confirming that in vitro autoprocessing of the 17-kDa form occurs at the C-terminus to yield the truncated forms. The 17-kDa form and the newly described 13-kDa form of proteinase were identified in virions collected from the rhesus monkey CMMT cell line chronically infected with M-PMV, confirming that multiple forms exist in vivo.

Substrates and inhibitors of human T-cell leukemia virus type 1 (HTLV-1) proteinase.

Human T-cell leukemia virus type 1 (HTLV-1) proteinase, a 125 residue polypeptide, was chemically synthesized using the solid phase method. The crude product was purified, renaturated and proteolytic activity was tested using oligopeptide substrates derived from processing sites of various retroviral polyproteins. Cleavage of the oligopeptide substrates together with an initial study using a series of HIV-1 and MAV (myeloblastosis associated virus) proteinase inhibitors suggest that the substrate specificity of HTLV-1 proteinase is very close to that of BLV (bovine leukemia virus) proteinase and distinct from that of both HIV-1 and MAV proteinases.

Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase.

We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.

Proteolytic processing of particle-associated retroviral polyproteins by homologous and heterologous viral proteinases.

Retroviral proteinase(PR)-catalyzed cleavage of the viral Gag and Gag-Pol polyproteins within the nascent virus particle is required for productive viral infection. Kinetic characterization and specificity analyses have been reported for several retroviral PR using oligopeptide substrates. In this study, we performed a comparative analysis of PR from avian, bovine, simian and human retroviruses using polyproteins of human immunodeficiency virus (HIV) type 1 or avian leukosis virus as substrates. Polyproteins were derived from immature virus-like particles purified from culture medium of transfected or recombinant baculovirus-infected cells. Specific cleavage to the correct size intermediate and end products occurred in the presence of detergent and homologous PR. HIV-1 PR cleaved its Gag precursor to completion at a concentration of approximately 25 nM but cleaved the Gag-Pol precursor incompletely even at fourfold higher PR concentration. In contrast to the requirement for high ionic strength for peptide cleavage reported previously, we found that Gag protein cleavage by HIV-1 PR proceeded best at low ionic strength, for both of the protein substrates tested. HIV-2 PR was approximately sixfold less active than HIV-1 PR. PR from avian myeloblastosis-associated virus (MAV) yielded efficient cleavage of the HIV-1 polyprotein only at concentrations above 1 microM. Both enzymes were stimulated by high salt and their cleavage products were identical or very similar to those of HIV-1 PR. A mutant of MAV PR engineered to cleave HIV-1 peptide substrates did not cleave the HIV-1 polyprotein at a concentration of 0.4 microM. The PR of Mason Pfizer monkey virus cleaved this polyprotein very poorly, whereas PR of bovine leukemia virus cleaved it, albeit at different sites.

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli.

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.