Diana L. Toledo

Viral DNA and a viral peptide can act as cofactors of adenovirus virion proteinase activity

HUMAN adenovirus (Ad2), like many other viruses1, contains a virion-associated proteinase essential for the synthesis of infectious virus particles2–4. We observed proteinase activity in wild-type virus but not in the ts-1 virus2, which contains a mutation in the Ad2 L3 endoprotease gene5 that confers temperature-sensitive processing of virion precursor proteins. Unexpectedly, we did not observe proteinase activity with purified recombinant6,7 endoprotease protein (Mr 23 K). Purified recombinant endo-protease protein, however, complemented the mutation in ts-1 virions, restoring proteinase activity when mixed together. This implied that cofactors may be required. Here we reconstitute proteinase activity in vitro with three purified viral components: (1) the recombinant endoprotease protein; (2) an 11-amino-acid peptide that originates from the carboxy terminus of pVI, the precursor to virion component VI; and (3) adenovirus DNA. The use of DNA for a proteinase activity is unprecedented.

[27] Omptin: An Escherichia coli outer membrane proteinase that activates plasminogen

Publisher Summary This chapter examines omptin, which is an Escherichia coli outer membrane proteinase that activates plasminogen. The new assays are based on the observations that omptin cleaves between basic amino acids and the aminopeptidases will not cleave after a blocked amino acid. Thus, the compounds benzyloxycarbonylarginylarginine 4-methyl-7-coumarylamide or Cbz-Arg-Arg-NHMec and Cbz-Ala-Lys-Arg-NHMec will not be cleaved by aminopeptidase M. In the presence of omptin, either substrate is cleaved to Arg-NHMec. When Arg-NHMec is incubated with aminopeptidase M, the highly fluorescent cleavage product aminomethylcoumarin is formed. This assay with synthetic, fluorogenic substrates is extremely sensitive. Tenfold less omptin is required in this assay to obtain a quantitative signal of enzyme concentration compared to that required in a plasminogen activation assay. The plasminogen activator activity of omptin is optimized with a two step assay. In the first step, omptin is incubated with plasminogen in a solution containing the variable for the experiment. After 5 min, the resultant plasmin is assayed by diluting the reaction mixture at least 10-fold in a solution containing the plasmin substrate (Ile-Pro-Arg-NH)2-rhodamine. The rate of hydrolysis of the plasmin substrate is then monitored under optimal conditions.

Temporal and spatial control of the adenovirus proteinase by both a peptide and the viral DNA

The adenovirus proteinase (AVP) uses both an 11-amino acid peptide (pVIc) and the viral DNA as cofactors to increase its catalytic rate constant 6000-fold. The crystal structure of an AVP-pVIc complex at 2.6-A resolution reveals a new protein fold of an enzyme that is the first member of a new class of cysteine proteinases, which arose via convergent evolution.

Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor

The type of proteinase and the nature of the active site of the human adenovirus proteinase are unknown. For these reasons we produced an inhibitor profile of the enzyme. Enzyme activity in disrupted virions was inhibited by several serine‐specific as well as cysteine‐specific proteinase inhibitors. Of the inhibitors that worked, the most useful potentially in illuminating the nature of the active site was bovine pancreatic trypsin inhibitor (BPTI), and for this reason we extensively characterized the interaction with BPTI. In disrupted virions, the enzyme is irreversibly inhibited by BPTI with a K i of 35 nM and a k i of 6.2 × 10−4 s−1. One reason enzyme activity is inhibited is that BPTI, a basic protein, precipitates the viral DNA, a cofactor of enzyme activity. In vitro with purified components, BPTI acts as a competitive inhibitor (K i 2 μM) of the recombinant proteinase complexed with its 11‐amino‐acid cofactor pVIc. The recombinant endoproteinase is heat labile whereas its 11‐amino‐acid cofactor is heat stable. We estimate there are about 50 molecules of proteinase per virus particle.

Plasminogen activators in human breast cancer cell lines: Hormonal regulation and properties

To understand the hormonal regulation of plasminogen activators (PAs) in human breast cancer, we have examined the hormonal regulation and properties of PAs in four human breast cancer cell lines that differ markedly in their estrogen receptor (ER) content: MCF-7 cells contain high levels of ER (approx 7 pmol/mg DNA) and their PA activity was increased 3-4-fold by physiological concentrations of estradiol; T47-D and ZR-75-1 cells contain lower levels of ER (0.9 and 2.1 pmol/mg DNA respectively) and their PA activity was also increased 3-4-fold by estradiol. In contrast, MDA-MB-231 cells, which do not contain ER, showed a high level of PA activity that was not modulated by estradiol. SDS-PAGE followed by zymography indicated that MCF-7 cells secreted tissue-type PA (t-PA), T47-D and ZR-75-1 cells secreted urokinase-type PA (u-PA), and MDA-MB-231 cells secreted both types of PAs. The types of PAs secreted by these cell lines did not change upon treatment with estradiol. Dose-response curves for the stimulation of MCF-7 PA activity by different estrogens showed an excellent correlation between affinities of the estrogens for ER and their potency in stimulating PA activity. With a clonal subline of MCF-7 cells, MCF-L, a soluble inhibitor of both t-PA and u-PA was secreted. Incubation of purified t-PA or u-PA with the serum-free conditioned medium from MCF-L cells resulted in a shift in the mobility of t-PA and u-PA in SDS-polyacrylamide gels to forms increased in molecular mass by about 50,000-70,000. The shifts in molecular mass could be prevented by the presence of the competitive inhibitor p-aminobenzamidine, indicating that the active sites of the PAs were involved in the formation of these complexes. Furthermore, co-cultivation, of RT4-D rat neuroblastoma cells, which exhibit high levels of t-PA activity, with MCF-L cells resulted in a marked decrease in the PA activity of the RT4-D cells. Our results were consistent with the following conclusions: t-PA, u-PA or both were secreted by human breast cancer cells. In the ER-containing cell lines, depending upon the specific cell line, t-PA or u-PA was stimulated by estrogens. The unstimulated levels of PA activity and the magnitude of PA stimulation by estrogens were not closely related to ER content.(ABSTRACT TRUNCATED AT 400 WORDS)