No abstract

The ␤-amyloid peptide (A␤) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as ␤-and ␥-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APPGal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cellconditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the ␥-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases A␤ generation and that, in cells stably expressing APP, the generation of A␤ is strongly decreased by the Src tyrosine kinase inhibitor PP2. ␤-Amyloid (A␤)1 deposition in the so-called amyloid plaques is one of the main features of Alzheimer's pathology. ␤-Amyloid consists of ϳ4-kDa peptides derived from the proteolytic processing of a membrane protein named amyloid precursor protein (APP). This amyloidogenic processing is driven by two enzyme activities, ␤-site APP cleaving enzyme (BACE) and ␥-secretase. BACE cleaves APP at 28 residues from the boundary between the extracellular/intraluminal domain of APP and the transmembrane domain of the protein (for a review see Ref. 1), releasing a large soluble protein, including nearly all the extracellular/intraluminal part of APP, and a short transmembrane peptide, including the 99 C-terminal residues of APP. This transmembrane C99 stub is a substrate for the ␥-secretase activity, which cleaves it, in a presenilin-dependent fashion, within the membrane ␣-helix, giving rise to the A␤ peptide 40 -42 amino acids long and to a peptide named APP intracellular domain (AID), which includes the small C-terminal cytosolic domain of APP (for a review see Refs. 2 and 3).The functions of APP and its proteolytic processing are still unknown. However, although the functions of the APP ectodomain remain elusive, there is increasing evidence that its cytodomain is the center of a complex network of interactions with several proteins, involved in vesicle transport and in signal transduction. In fact, it was demonstrated that APP cytoplasmic domain interacts with kinesin light chain and contributes to vesicles transport (4), thus suggesting that APP cleavage could regulate the transport of vesicles in the axons. On the other hand, the APP cytodomain binds several PTB domain-containing pro-

Heavy metals can cause several genotoxic effects on cells, including oxidative stress, DNA sequence breakage and protein modification. Among the body organs, skin is certainly the most exposed to heavy metal stress and thus the most damaged by the toxic effects that these chemicals cause. Moreover, heavy metals, in particular nickel, can induce the over-expression of collagenases (enzymes responsible for collagen degradation), leading to weakening of the skin extracellular matrix. Plants have evolved sophisticated mechanisms to protect their cells from heavy metal toxicity, including the synthesis of metal chelating proteins and peptides, such as metallothioneins and phytochelatins (PC), which capture the metals and prevent the damages on the cellular structures. To protect human skin cells from heavy metal toxicity, we developed a new cosmetic active ingredient from Lycopersicon esculentum (tomato) cultured stem cells. This product, besides its high content of antioxidant compounds, contained PC, effective in the protection of skin cells towards heavy metal toxicity. We have demonstrated that this new product preserves nuclear DNA integrity from heavy metal damages, by inducing genes responsible for DNA repair and protection, and neutralizes the effect of heavy metals on collagen degradation, by inhibiting collagenase expression and inducing the synthesis of new collagen.

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of Rab-GAP1, and EF1␣, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays. Molecular & Cellular Proteomics 6:333-345, 2007.

Thanks to these activities, the Rubus idaeus liposoluble extract has several potential applications in skin care cosmetics: it can be used as hydrating and moisturizing ingredient in face and body lotions, and as anti-ageing product in face creams specifically designed to fight wrinkle formation.