α-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its α-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.

Platinum-complexes represent some of the most successful groups of clinically used anticancer drugs. Their mechanism of action relies on the formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7) and involving one or two spatially close residues. The formation of stable DNA adducts is recognized as a DNA damaging event and, ultimately, drives cells to death. Nevertheless, nucleobases are not the only reliable targets of these drugs and other biomolecules can be involved. Among them large interest has been devoted to proteins since they contain several potential reactive sites for platinum (His, Met, and Cys) and, in particular, because the reaction of the metal with sulfur containing groups is a kinetically favored process. As a result, the occurrence of protein adducts and DNA-protein cross-links must be further taken into account in order to fully define cisplatin mechanism of action. Herein, we will summarize the most recent experimental evidence collected so far on protein-cisplatin adduct formation to better dissect its correlation with the drug pharmacological profile. Indeed, in addition to modulation of drug bioavailability and toxicity, the potential role of proteins as reaction intermediates or reservoir systems in platinum drugs can be envisaged. Additionally, the effects of Pt-coordinating groups on the chemical reactivity of the metal complexes will be reviewed. From all these outcomes a general model for Pt-based drugs mechanism of action can be drawn which is more articulate than the one currently supported. It claims proteins as reactive intermediates for DNA platination and it defines them as relevant to fully describe the clinical potential of this class of anticancer drugs.

The mechanism of action of clinically used Pt-based drugs is through the formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7) and involving one or two spatially closed residues. Nevertheless, proteins can represent alternative targets since in particular sulphur groups, present in cysteine or methionine residues, can efficiently coordinate platinum. Here we have characterized the reactivity profile of cisplatin, transplatin and of two trans-platinum amine derivatives (TPAs) towards three different proteins, bovine α-lactalbumin (α-LA), hen egg lysozyme (LYS) and human serum albumin (HSA). Our results demonstrate that generally the tested metal complexes react with the selected target causing protein oligomerization, likely through a cross-linking reaction. Interestingly, the extent of such a process is largely modulated by the target protein and by the chemical features of the metal complex, TPAs being the most efficient platinating agents. From a structural point of view the resulting reaction products turned out to be dependenting on the nature of the metal complexes. However, in all instances, a transfer reaction of the metal complex to DNA can also occur, maintaining the relevance of nucleic acids as a biological target. These results can be used to better rationalize the different pharmacological profiles reported for cisplatin and TPAs and can help in designing more predictive SARs within the series.

No abstract

AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that circularization is not required for the bactericidal activity, but it is important to stabilize the native structure. Moreover, it is possible to reduce the sequence to a minimal AS-48 domain without causing inactivation of this bacteriocin.