SynopsisSince ancient times, human beings have been trying to protect their skin against the adverse effects of the sun. From the first mineral sunscreens used by Egyptians, to the current more sophisticated ultraviolet (UVA/UVB) organic sunscreens, progress has been made in terms of sun protection and deeper knowledge of skin physiology has been acquired in the process. The solar spectrum is composed of radiations of various wavelengths having specific, as well as overlapping effects on skin. UVB is mainly responsible for sunburn and DNA dimer formation that can lead to mutation. UVA generates oxidative reactions affecting DNA, proteins and lipids, and is also immunosuppressive. Recently, visible light and infrared radiation (IR) have been associated with oxidative damage and IR has been additionally linked to adverse heat effects on skin. Numerous other extrinsic factors, related to environment and lifestyle, also affect the appearance of skin, precipitating ageing. New molecular mechanisms linking sun and environmental factors to skin ageing have been identified: IR affects mitochondrial integrity and specific heat receptors also mediate some of its effects, tryptophan is a chromophore for UVB, and the aryl hydrocarbon receptor (AhR) is activated by light and xenobiotics to alter skin physiology. Integrating all these new elements is changing the way we think about skin extrinsic ageing. Is UVA/UVB sunscreen protection still enough for our skin?R esum e Depuis les temps anciens, lesêtres humains ont essay e de prot eger leur peau contre les effets n efastes du soleil. Depuis les ecrans minraux d'abord utilis es par les Egyptiens, aux filtres solaires organiques sophistiqu es actuels (UVA/UVB), des progr es ont et e r ealis es en termes de protection contre le soleil, et une connaissance approfondie de la physiologie cutan ee a et e acquise durant ce temps. Le spectre solaire est compos e de radiations de longueurs d'onde diff erentes poss edant des effets sp ecifiques, ainsi que des effets redondants sur la peau. Les UVB sont les principaux responsables des coups de soleil et de la formation de dim eres d'ADN qui peuvent conduire a une mutation. Les UVA g en erent des r eactions d'oxydation qui affectent l'ADN, des prot eines, et les lipides, et agissent egalement comme immunosuppresseur. R ecemment, les rayonnements de la lumi ere visible et de l'infrarouge (IR) ont et e associ es a des dommages oxydatifs, et l'IR a et e en outre li e a des effets ind esirables de la chaleur sur la peau. De nombreux autres facteurs extrins eques li es a l'environnement et au mode de vie affectent aussi l'aspect de la peau, acc el erant le vieillissement. De nouveaux m ecanismes mol eculaires reliant le soleil et les facteurs environnementaux au vieillissement de la peau ont et e identifi es: l'IR affecte l'int egrit e mitochondriale; des r ecepteurs thermiques sp ecifiques servent egalement de m ediateur de certains de ses effets; le tryptophane est un chromophore UVB; le r ecepteur d'hydrocarbures aryles (AhR) est activ e p...

The actin cytoskeleton is involved in numerous cellular functions such as cell motility, mitogenesis, morphology, muscle contraction, cytokinesis, and establishment of cell polarity. The members of the Rho subfamily of small GTP-binding proteins emerge as key regulators of cytokeleton organization. Rho, Rac, and CDC42 are implicated in the regulation of actin microfilament organization of different cell structures, such as stress fibers linked to focal adhesions and membrane ruffles induced by extracellular stimuli. Rho proteins also regulate the activity of several enzymes involved in the formation of phospholipid derivatives, which could mediate their effect on the cytoskeleton. The activity of Rho proteins is regulated by many nucleotide exchange factors and GTPase-activating proteins, some of which are oncogene products, and other disease-associated proteins. The potential role of these small GTP-binding proteins in carcinogenesis is suggested by the actin reorganization seen in transforming cells and by the need for functional Rho proteins in Ras mitogenic activation.

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.