An analytical model for the phase behavior of cholesteryl esters in intracellular inclusions.

The cholesteryl ester-rich, intracellular inclusions that characterize atherosclerotic plaque are capable of existing in a metastable, relatively fluid state for long periods of time. We have developed an analytical model which explains this metastability, and other aspects of the phase behavior, of physiologically relevant, phospholipid-stabilized dispersions of cholesteryl ester mixtures. The model, based on classical nucleation theory, incorporates temperature, time and lipid composition as independent variables. Differential scanning calorimetry was used to elucidate the model. The dispersions consisted of cholesteryl palmitate and an ester containing a long-chain, unsaturated or polyunsaturated, fatty acid. When a dispersion of approx. 1-microns droplets is melted, then cooled, crystallization is preceded by the formation of small crystalline nuclei (homogeneous nucleation). Nucleation is energetically unfavorable until (typically) well below the melting point. sigma, the tension between the surface of the crystal nucleus and surrounding fluid, is a measure of the difficulty in forming nuclei. This parameter was found to increase with the content of unsaturated ester. sigma was found to increase with increasing triacylglycerol content, and to decrease upon addition of free cholesterol.

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.

Purification of active herpes simplex virus-1 protease expressed in Escherichia coli.

Assembly of viral capsids for replication of herpes simplex virus requires the proteolytic processing of the assembly protein ICP35. The protease responsible for this process is encoded within the 635-amino acid open reading frame of the UL26 gene of the virus. A simple purification scheme is given in this report for the native, mature form of the protease expressed in Escherichia coli. The scheme allows the preparation of milligram quantities of purified enzyme for elucidation of kinetic mechanism as well as for structural studies. Utilizing a 13-residue peptide substrate representing the natural cleavage site that releases the protease, kcat and Km values of the purified native enzyme are 2.0 min-1 and 0.88 mM, respectively. Thus, peptide cleavage is less efficient than reported for other viral proteases. The possibility exists that viral or cellular factors are involved in vivo for activation of the protease for herpes capsid maturation.

Peptide substrate cleavage specificity of the human cytomegalovirus protease.

The human cytomegalovirus UL80 gene encodes an 80-kDa precursor polyprotein whose N-terminal 256-amino acid domain is a protease. This enzyme cleaves a specific peptide bond that results in its own release from the precursor, as well as a peptide bond near the C terminus of the viral assembly protein. The latter cleavage is apparently required for encapsidation of the viral genomic DNA and maturation of the viral capsid. A series of peptide substrates, representing the assembly protein cleavage site, was used to study the enzyme's substrate requirements and specificity. It was found that efficient cleavage minimally required the amino acid residues spanning the P4 to P4' positions. Substitution at any of these residues adversely affected the reaction. Conservation of the hydrophobic residues at P3 and P4 was essential. In addition, cleavage of a peptide representing the protease domain release site was reduced almost 100-fold relative to cleavage of the assembly protein maturation site peptide substrate.