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 arisein vitroby 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 thatin vitroautoprocessing 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 existin vivo

Simple Method for Screening Candida Species Isolates for the Presence of Secreted Proteinases: a Tool for the Prediction of Successful Inhibitory Treatment

The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans, C. tropicalis, and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition

Analysis of Autoprocessing of Mason-Pfizer Monkey Virus Proteinase in Vitro Three Active Forms of Proteinase

Mason-Pfizer Monkey Virus (M-PMV, also called SRV-3) is a primate retrovirus that was first isolated from a spontaneous breast carcinoma of a female rhesus monkey Macaca mulatta.1,2 Infection of macaque species with M-PMV causes an AIDS-like disease.3,6 M-PMV is characterized by the self-assembly of the Gag, Gag-Pro and Gag-Pro-Pol precursors into intracytoplasmic particles (procapsids) within the infected cell cytoplasm. These morphogenetic properties of the virus are typical for D-type retrovirus.7,

Δ12-Fatty Acid Desaturase from <i>Candida parapsilosis</i> Is a Multifunctional Desaturase Producing a Range of Polyunsaturated and Hydroxylated Fatty Acids

<div><p>Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named <i>Cp</i>Fad2 and <i>Cp</i>Fad3, respectively, from the opportunistic pathogenic yeast <i>Candida parapsilosis</i>. Overexpression of <i>Cp</i>Fad3 in <i>Saccharomyces cerevisiae</i> strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, <i>i.e.</i>, α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). <i>Cp</i>Fad2 produces a range of Δ12- and Δ15-PUFAs. The major products of <i>Cp</i>Fad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of <i>Cp</i>Fad2. These results demonstrate that <i>Cp</i>FAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina).</p></div

Relative abundance of fatty acids in FAME extracts from <i>C. parapsilosis</i>.

<p>The relative amount of fatty acids is expressed as a percentage of total fatty acid methyl esters. Values represent means of three cultivations ± standard deviation. n.d.: FAME not detected.</p

Relative abundances of fatty acids in FAME extracts of total cellular lipids from yeast strains.

<p>The relative amount of fatty acids is expressed as a percentage of total fatty acid methyl esters. Strains supplemented with 0.5 mM linoleic acid and 1% tergitol are marked with “+”. Values represent means of three cultivations ± standard deviation. n.d.: FAME not detected.</p

Relative abundances of TMS derivatives of hydroxy FAs in FAME extracts from <i>Cp</i>FAD2, <i>Cp</i>FAD3 and Empty strains.

<p>The relative amount of FAME-TMS derivatives is expressed as a percentage of total fatty acid methyl esters. Strains supplemented with 0.5 mM linoleic acid and 1% tergitol are marked with “+”. Values represent means of three cultivations ± standard deviation. n.d.: TMS-derivative not detected.</p

GC chromatograms of FAME extracts from yeasts supplemented with hexadecadienoic acid (Δ9,Δ12-16:2).

<p>GC/MS analysis of FAME extracts from (<b>A</b>) Empty strain supplemented with hexadecadienoic acid (Δ9,Δ12-16:2), (<b>B</b>) <i>Cp</i>FAD2 strain, (<b>C</b>) <i>Cp</i>FAD2 strain supplemented with Δ9,Δ12-16:2 and (<b>D</b>) <i>Cp</i>FAD3 strain supplemented with Δ9,Δ12-16:2. Hexane containing internal standard (hexadecane at concentration of 20 μg/ml) was used in preparation of all extracts.</p