Biochemical characterization of Ty1 retrotransposon protease

Abstract

Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His 6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.

Functional Study of the Retrotransposon-Derived Human PEG10 Protease

“…In addition, the D-S-G-A active site motif of human PR PEG10 is also identical to that of human Ddi1 and Ddi2 PRs, but this consensus motif is followed by a Ser residue (D-S-G-A-S) in PR PEG10 (Figure 1). Ser in this position is not characteristic of retroviral or Ddi1/Ddi2 PRs [29] but can be found in the Ty1 retrotransposon PR [30]. The D-T/S-G-A active site motif of most retroviral PRs is followed by an Asp residue, but in contrast with this Asp residue, a Ser in this position does not enable salt-bridge formation between PR PEG10 subunits.…”

“…Our data imply low catalytic efficiency of PR PEG10 , but detailed comparison cannot be made due to the limited data on the catalytic efficiencies of retroviral-like proteases. To date, the proteolytic activity of a recombinant protein was proven in vitro only for Leishmania major Ddi1 [33] and Saccharomyces cerevisiae Ty1 retroviral-like proteases [30], while catalytic efficiency was determined only for the latter one. The structural characteristics of PR PEG10 indicate high similarity with other retroviral-like proteases; thus, the dimer stability of PR PEG10 is potentially lower than that of most retroviral proteases [31], and is more comparable to that of retroviral-like proteases.…”

“…The structural characteristics of PR PEG10 indicate high similarity with other retroviral-like proteases; thus, the dimer stability of PR PEG10 is potentially lower than that of most retroviral proteases [31], and is more comparable to that of retroviral-like proteases. For example, Ty1 PR was found to possess trans activity and has very low specific activity as compared to retroviral proteases (e.g., HIV-1, HTLV-1, BLV, and MMLV) [30]. Due to the limited data, future studies need to reveal whether all human retroviral-like proteases (including PEG10, Ddi1, and Ddi2) have lower specific activity than retroviral proteases.…”

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

“…Dimer stability of GST-SASP14 was investigated ( Figure 5 ) by determination of the urea concentration causing 50% loss of enzyme activity (also referred as urea dissociation constant, UC 50 ). The obtained stability values were compared with the values reported previously for HIV-1, XMRV [ 28 ], and Ty1 retrotransposon proteases [ 37 ].…”

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates

“…The enzyme activity measurements were performed by using a recombinant fluorescent protein substrate-based protease assay, which has been previously applied successfully to study TEV and HIV-1 proteases [ 23 ], the retroviral-like protease of human paternally expressed gene 10 (PEG10) protein [ 35 ] and yeast retrotransposon Ty1 [ 36 ]. The previously developed method was slightly modified and some improvements were introduced as follows.…”