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C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host’s immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host’s environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation, principally in the central nervous system (CNS), of the abnormal form (PrPTSE) of a host encoded protein, the cellular prion protein (PrPC). In humans some forms of TSEs have a genetic etiology, as they are associated to mutations in the PrP gene: thus it is extremely important to elucidate the role played by the altered residue in the pathological conversion of the protein. Aim of our work was to deduce the involvement of the mutated residue in the molecular mechanisms underlying genetic TSE pathogenesis by means of a quantitative mass spectrometry analysis of the PrPTSE allotypes accumulated in the brains of genetic Creutzfeldt-Jakob disease (gCJD) affected individuals. We have integrated our previous data on the gCJD patient carrying the R208H mutation with those regarding V210I gCJD patients in order to verify if diverse mutations regulate in a different fashion the process of molecular pathogenesis. The LC/MS (liquid chromatography/mass spectrometry) protocol developed by us (Schininà M.E. et al., 2003. Pure Appl. Chem., 75: 317-323) has been implemented in order to set up, by means of appropriate synthetic deuterated (internal standards) and non-deuterated (calibrants) peptides, a quantitative analysis of the pathological PrP allotypes. In all cases the amount of the mutant allotype is higher than the wild-type counterpart: in the R208H subject the mutant/wild-type ratio has been again estimated and it was around 3/1, while in all the V210I individuals it was lower than 2/1. These results demonstrate that the presence of the mutation is partially involved in favouring the pathological conversion of PrP, but it is not the only factor implicated in the pathogenesis of TSEs as the ratio of the two allotypes does not reveal a significantly higher amount of the mutant allotype compared to the wild-type one. Further investigations are needed in order to elucidate the molecular mechanisms underlying the pathogenesis of genetic human TSEs.

The design of chimeric proteins is a major held of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors. (C) 2000 Academic Press.

Several proteins are covalently bound to the cell wall glucan (glucan-associated proteins (GAPs)) in Candida albicans and different drugs may cause their modulation. Proteomic analysis is a suitable approach to study differential GAP patterns between control and drug-treated cells. Since antimycotics induce variation in GAP content, we investigated the effect of a sublethal dose of micafungin and observed a clear increase in Bgl2p, an enzyme with glucanosyltransferase activity, with respect to a general decrease in cell wall protein content. Immunoelectron microscopy using mouse antiserum confirmed this increase of Bgl2p on the outer cell wall but also revealed a dramatic increase in the immature Bgl2p isoform in the cytoplasm of drug-treated cells. Since this increased expression of Bgl2p is clearly dependent upon micafungin treatment, this enzyme appears to be one of the survival strategies of C. albicans and thus could be considered the molecular basis of antifungal resistance and also as a potential valuable candidate for future vaccine development.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37°C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological vs. physiological conditions.

The amino acid sequences of two pyridoxal phosphate-dependent enzymes, namely glutamate decarboxylase and 4-aminobutyrate aminotransferase, were solved by a combination of direct protein sequencing and cDNA sequencing. In the case of glutamate decarboxylase from Escherichia coli, correct ordering of three internal peptides was achieved by sequencing a fragment of DNA generated by PCR. In the case of 4-aminobutyrate aminotransferase from pig liver, chemical studies on the reaction of the enzyme with a suicide inhibitor allowed a detailed description of the mechanism of inactivation and collection of partial sequence information on the protein. The sequence of the protein was completely reconstructed after analysis of further peptide fragments derived by proteolytic and chemical cleavages, with the help of the information from the cDNA-deduced sequence of pig brain 4-aminobutyrate aminotransferase performed by another group (Kwon, Park and Churchich [1992] J. Biol. Chem. 267, 7215-7216).

A nuclear glycoprotein with an apparent Mr of 66,000 Da has been isolated from pig kidney chromatin after extraction with urea, guanidine-HCl and 2 M NaCl, and some of its structural features have been characterized. It belongs to the group of N-glycosylated proteins, which in the nucleus has so far received little attention. From its monosaccharide composition and recognition by lectins its oligosaccharides appear to be of high mannose and/or hybrid types. Some properties of its protein moiety suggest that it has a role in the packing of the DNA loops in the condensed chromatin.

The complete primary structure of saporin SO-6, a ribosome-inactivating protein extracted from Saponaria officinalis seeds, has been determined. The sequence was reconstructed following purification and analysis of peptides obtained after digestion of the protein with different proteolytic agents. The protein is composed of 253 amino acids, corresponding to a molecular weight of 28,621 Da. Comparison of the primary structure of SO-6 with the sequence deduced from cDNA, shows amino acid substitutions in 11 positions, suggesting a tissue-related genetic variability. When the sequence of saporin is compared to those of two related proteins, ricin A chain and trichosanthin, a low degree of similarity (12%) is found; nevertheless some considerations about structure-function relationships and evolution of RIPs are possible.