Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.
The specificities of the proteases of 11 retroviruses representing each of the seven genera of the family Retroviridae were studied using a series of oligopeptides with amino acid substitutions in the P2 position of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr2Pro-Ile-Val-Gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (HIV-1). This position was previously found to be one of the most critical in determining the substrate specificity differences of retroviral proteases. Specificities at this position were compared for HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus, MasonPfizer monkey virus, mouse mammary tumor virus, Moloney murine leukemia virus, human T-cell leukemia virus type 1, bovine leukemia virus, human foamy virus, and walleye dermal sarcoma virus proteases. Three types of P2 preferences were observed: a subgroup of proteases preferred small hydrophobic side chains (Ala and Cys), and another subgroup preferred large hydrophobic residues (Ile and Leu), while the protease of HIV-1 preferred an Asn residue. The specificity distinctions among the proteases correlated well with the phylogenetic tree of retroviruses prepared solely based on the protease sequences. Molecular models for all of the proteases studied were built, and they were used to interpret the results. While size complementarities appear to be the main specificity-determining features of the S2 subsite of retroviral proteases, electrostatic contributions may play a role only in the case of HIV proteases. In most cases the P2 residues of naturally occurring type 1 cleavage site sequences of the studied proteases agreed well with the observed P2 preferences.
A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.
The protease (PR) of Murine leukemia virus (MLV) was expressed in Escherichia coli, purified to homogeneity and characterized by using various assay methods, including HPLC-based, photometric and fluorometric activity measurements. The specificity of the bacterially expressed PR was similar to that of virion-extracted PR. Compared with human immunodeficiency virus type 1 (HIV-1) PR, the pH optimum of the MLV enzyme was higher. The specificity of the MLV PR was further compared with that of HIV-1 PR by using various oligopeptides representing naturally occurring cleavage sites in MLV and HIV-1, as well as by using bacterially expressed proteins having part of the MLV Gag. Inhibitors designed against HIV-1 PR were also active on MLV PR, although all of the tested ones were substantially less potent on this enzyme than on HIV-1 PR. Nevertheless, amprenavir, the most potent inhibitor against MLV PR, was also able to block Gag processing in MLV-infected cells. These results indicate that, in spite of the similar function in the life cycle of virus infection, the two PRs are only distantly related in their specificity.
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