Two small, cysteine-rich and cationic antifungal proteins from Penicillium chrysogenum: A comparative study of PAF and PAFB.

The filamentous fungus Penicillium chrysogenum Q176 secretes the antimicrobial proteins (AMPs) PAF and PAFB, which share a compact disulfide-bond mediated, ß-fold structure rendering them highly stable. These two AMPs effectively inhibit the growth of human pathogenic fungi in micromolar concentrations and exhibit antiviral potential without causing cytotoxic effects on mammalian cells in vitro and in vivo. The antifungal mechanism of action of both AMPs is closely linked to - but not solely dependent on - the lipid composition of the fungal cell membrane and requires a strictly regulated protein uptake into the cell, indicating that PAF and PAFB are not canonical membrane active proteins. Variations in their antifungal spectrum and their killing dynamics point towards a divergent mode of action related to their physicochemical properties and surface charge distribution. In this review, we relate characteristic features of PAF and PAFB to the current knowledge about other AMPs of different sources. In addition, we present original data that have never been published before to substantiate our assumptions and provide evidences that help to explain and understand better the mechanistic function of PAF and PAFB. Finally, we underline the promising potential of PAF and PAFB as future antifungal therapeutics.

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli.

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45°C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17±2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.

Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole.

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.

A novel sequence element is involved in the transcriptional regulation of expression of the ERG1 (squalene epoxidase) gene in Saccharomyces cerevisiae.

Squalene epoxidase is an essential enzyme in the ergosterol-biosynthesis pathway. It catalyzes the epoxidation of squalene to 2,3-oxidosqualene and is the specific target of the antifungal drug terbinafine. Treatment of yeast cells with this inhibitor leads to squalene accumulation and sterol depletion. As ergosterol fulfils several essential functions, each requiring optimal sterol concentrations, synthesis of sterols in yeast must be tightly regulated. This study focuses on the sterol-mediated regulation of expression of the ERG1 gene, which codes for squalene epoxidase in Saccharomyces cerevisiae. Inhibition of ergosterol biosynthesis with terbinafine increases the expression of ERG1 in a concentration-dependent manner to a maximum of sevenfold. Inhibition of later steps in the ergosterol-biosynthetic pathway by ketoconazole, an inhibitor of the lanosterol-14alpha-demethylase, and U18666A, an inhibitor of the squalene-2,3-epoxide-lanosterol cyclase, also induce expression of ERG1, suggesting that ERG1 expression is positively regulated by diminished intracellular ergosterol levels. The regulatory effect of sterols is manifested at the level of transcription. Deletion analysis of the ERG1 promoter identified a novel regulatory DNA sequence element. Two 6-bp direct repeats, separated by 4 bp, AGCTCGGCCGAGCTCG, are unique to the ERG1 promoter. A DNA fragment containing this region confers ergosterol-regulated expression on an otherwise unregulated CYC1 promoter construction. One copy of the 6-bp element, AGCTCG, is sufficient to confer regulation, albeit less effectively than when both elements are present, whereas the removal of both elements from the ERG1 promoter leads to the loss of sterol-dependent ERG1 regulation.

Targeting of aminopeptidase I to the yeast vacuole is mediated by Ssa1p, a cytosolic member of the 70-kDa stress protein family.

The two cytosolic members of the highly conserved 70-kDa stress protein family, Ssa1p and Ssa2p, were specifically retained by the prepro-NH(2) extension of the vacuolar aminopeptidase I precursor (pAPI) conjugated to agarose (Sulfolink). A temperature-sensitive mutant strain a1(ts)a234 (ssa1(ts) ssa2 ssa3 ssa4), when incubated at the restrictive temperature, was able to assemble the API precursor into dodecamers, but failed to pack pAPI into vesicles and to convert it into mature API (mAPI), a process that occurs in the vacuole. Altogether these results indicate that Ssa1p mediates the targeting of pAPI to the vacuole.

Equine SCID: mechanistic analysis and comparison with murine SCID.

V(D)J rearrangement is the molecular mechanism by which an almost limitless number of unique immune receptors is generated. V(D)J rearrangement involves two DNA breaks and religations resulting in two DNA joints; coding and signal joints. If V(D)J recombination is impaired (as in murine SCID (C.B-17 mouse] or RAG [Recombinase Activating Genes) deficient mice), B lymphocyte and T lymphocyte development is blocked and severe immunodeficiency results. The first animal model of SCID was reported in Arabian foals in 1973. Recently we demonstrated that the mechanistic defect in SCID foals is V(D)J recombination. However, the impairment of V(D)J recombination in SCID foals is phenotypically distinct from SCID mice in that both signal and coding joint ligation are impaired. Furthermore, though equine SCID and murine SCID have definite phenotypic differences, both defects are likely to be the result of defective expression of the catalytic subunit of the DNA-dependent protein kinase.

Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase.

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.

Export of steryl esters from lipid particles and release of free sterols in the yeast, Saccharomyces cerevisiae.

Fatty acyl esters of the yeast specific sterol, ergosterol, are exclusively stored in lipid particles. Under conditions of sterol deficiency, e.g., in the presence of terbinafine, an inhibitor of fungal squalene epoxidase, steryl esters are hydrolyzed, and sterols are set free for membrane formation. Lipid particles do not contain steryl-ester hydrolase activity themselves; the highest specific activity of this enzyme is found in the plasma membrane. Therefore, steryl esters have to be exported from lipid particles to their site of hydrolytic cleavage. This process of translocation and metabolic conversion was studied in vivo. Addition of nocodazole to terbinafine-treated cells did not disturb the mobilization of steryl esters, indicating that this process is not mediated by microtubuli-dependent vesicle flux. Under the influence of inhibitors of cellular energy production (azide and fluoride) and protein biosynthesis (cycloheximide) mobilization of steryl esters came to an halt. These results support the view that ongoing membrane proliferation may be a driving force for the release of sterols from steryl esters of lipid particles.

Characterization of lipid particles of the yeast, Saccharomyces cerevisiae.

Lipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed. The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment. Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins. Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments. Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively. The 43 kDa protein reacts with an antiserum against human apolipoprotein AII. In lipid particles of the yeast mutant strain S. cerevisiae erg6, which is deficient in sterol delta 24-methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol delta 24-methylation is associated with lipid particles. A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.

Degradation of glomerular basement membrane in diabetes. I. Susceptibility of diabetic and nondiabetic basement membrane to proteolytic degradation of isolated glomeruli.

Degradation of glomerular basement membrane in diabetic and nondiabetic rats was measured by incubating isolated basement membrane with a homogenate of glomeruli obtained from metabolically healthy rats. When diabetic basement membrane was used, there was a marked decrease in the amount of collagen-typical (hydroxyproline, hydroxylysine, glycine) and noncollagen-typical amino acids (proline, lysine, leucine) released in the supernatant of the incubation assay. A negative correlation was found between the amount of collagen-typical amino acids released by diabetic basement membrane and the duration of diabetes. The results indicate that the collagenous and noncollagenous peptides of diabetic basement membrane are less susceptible to proteolytic degradation than those of nondiabetic controls. This may be due to increased nonenzymatic glycosylation of diabetic basement membrane.

Degradation of glomerular basement membrane in diabetes. II. Proteolytic activity of diabetic and nondiabetic glomeruli.

The proteolytic effects of isolated glomeruli of diabetic rats on glomerular basement membrane of nondiabetic rats was investigated at various times after streptozotocin injection. One week after induction of diabetes, proteolytic activity remained unchanged as compared with nondiabetic controls. However, 4 and 10 weeks after streptozotocin injection, glomerular degradation of collagenous (but not noncollagenous) peptides of basement membranes increased (+24% as compared with control experiments). Using diabetic basement membrane as substrate, degradation of collagenous and noncollagenous peptides caused by diabetic glomeruli was 2.6-fold and 1.7-fold higher than in control experiments with nondiabetic glomeruli. The results indicate that the disturbed degradation of glomerular basement membrane in diabetes is not due to a decreased activity of glomerular proteolytic enzymes. In contrast, it can be concluded that the increased resistance of diabetic basement membrane to proteolytic degradation could be partially compensated by quantitative and qualitative changes of the proteolytic potential of diabetic glomeruli.