The protective effect of a novel sunscreen against blue light.

BACKGROUND AND OBJECTIVE: Premature skin ageing, and skin hyperpigmentation are influenced by exogenous factors, such as ultraviolet radiation and blue light. In this study, we assess the protective effect of a sunscreen (TDF® Blu Voile Sunscreen) in protecting the skin against the harmful effects of blue light irradiation in vivo and through the in situ quantitative and qualitative evaluation of protein carbonylation in human skin explants. METHODOLOGY: The protective effect of the test product against blue light was first evaluated ex vivo on human skin explants. The treated and non-treated explants were exposed to 14 J/cm2 of blue light 460 nm following which the protein carbonylation was evaluated by in situ epifluorescence imaging and separation by high-resolution gel electrophoresis. To determine whether the test product could also protect against the immediate and persistent pigmenting effect of blue light, two randomized in vivo studies were conducted, which included respectively 17 subjects with a skin phototype of IV and V (Fitzpatrick classification) and 22 subjects with a skin phototype of IV, V, and VI (Fitzpatrick classification). The duration of the study for each subject was 2 days (D1 and D2) for immediate observations and 5 days (D1-D5) for persistent observations. Specific zones on the subjects' back were either left non-treated or treated with the test product and were then exposed to a unique dose of blue light 415 nm. The onset of pigmentation between the treated and exposed zones was then assessed relative to the non-exposed treated zone through colorimetric measurements of the Individual Typology Angle (ITAo ). RESULTS: Human skin explants treated with test product showed significantly lower levels of accumulated carbonylated proteins, with a protection of 82%, following exposure to blue light 460 nm. Findings of the in vivo studies also indicated that the test product presented significantly better protective efficacy against immediate and persistent pigmentation induced by blue light 415 nm. CONCLUSION: Hence, it can be concluded that the test product can protect against the oxidative stress as well as the immediate and persistent pigmentation induced by blue light.

CONTEXTE ET OBJECTIF: Le vieillissement prématuré de la peau et l'hyperpigmentation cutanée sont influencés par des facteurs exogènes, tels que les rayons ultraviolets et la lumière bleue. Dans cette étude, nous évaluons l'effet protecteur d'un écran solaire (TDF® Blu Voile Sunscreen) en matière de protection de la peau contre les effets nocifs de l'irradiation à la lumière bleue in vivo et par l'évaluation quantitative et qualitative in situ de la carbonylation des protéines dans des explants cutanés humains. MÉTHODOLOGIE: L'effet protecteur du produit testé contre la lumière bleue a d'abord été évalué ex vivo sur des explants cutanés humains. Les explants traités et non traités ont été exposés à 14 J/cm2 de lumière bleue à 460 nm, après quoi la carbonylation des protéines a été évaluée par imagerie par épifluorescence in situ et séparation par électrophorèse sur gel à haute résolution. Afin de déterminer si le produit testé pouvait également protéger contre la pigmentation immédiate et persistante dues à lumière bleue, deux études in vivo randomisées incluant respectivement 17 sujets ayant un phototype cutané IV et V (classification de Fitzpatrick) et 22 sujets ayant un phototype cutané IV, V et VI (classification de Fitzpatrick) ont été menées. La durée de l'étude pour chaque sujet était de 2 jours (J1 et J2) pour les observations immédiates et de 5 jours (J1 à J5) pour les observations persistantes. Des zones spécifiques du dos des sujets ont été laissées non traitées ou bien traitées avec le produit testé, et ont ensuite été exposées à une dose unique de lumière bleue à 415 nm. L'apparition de la pigmentation entre les zones traitées et exposées a ensuite été évaluée par rapport à la zone traitée non exposée par des mesures colorimétriques de l'angle typologique individuel (Individual Typology Angle, ITAo). RÉSULTATS: Les explants cutanés humains traités avec le produit testé ont montré des taux significativement plus faibles de protéines carbonylées accumulées, avec une protection de 82 %, après une exposition à la lumière bleue à 460 nm. Les résultats des études in vivo ont également indiqué que le produit testé présentait une efficacité protectrice significativement meilleure contre la pigmentation immédiate et persistante induite par la lumière bleue à 415 nm. CONCLUSION: Par conséquent, on peut conclure que le produit testé peut protéger contre le stress oxydatif ainsi que contre la pigmentation immédiate et persistante induite par la lumière bleue.

Oxidative damage prevention in human skin and sensory neurons by a salicylic acid derivative.

BACKGROUND: Increased protein carbonylation is a hallmark of oxidative stress, protein homeostasis dysregulation and aging in the nervous system and skin. Sensory neurons interact with skin cells and are involved in skin homeostasis. We have previously reported that the 5-octanoyl salicylic acid (C8-SA), a salicylic acid derivative, increased C. elegans lifespan and delayed the accumulation of carbonylated proteins, through the stimulation of autophagy. OBJECTIVES: In this study we aimed to investigate if C8-SA protects human sensory neurons and human skin from extrinsic oxidative stressors as an approach to delay skin aging. METHODS: In vitro reconstituted human epidermis innervated with hiPSc-derived human sensory neurons, as well as ex vivo human organotypic full skin models were used. The fully differentiated sensory neurons were pretreated with C8-SA before oxidative stress induction. Skin explants were maintained in culture and treated topically with C8-SA before the application of urban pollutants. Carbonylated proteins were detected using amino-oxy functionalized fluorophores and quantified. Chaperone mediated autophagy was monitored with LAMP2A immunofluorescence. Inflammation, ROS detoxification and autophagy were assessed by RT-PCR. RESULTS: C8-SA prevented the accumulation of carbonylated proteins, both in human sensory neurons and skin explants. C8-SA stimulated chaperone-mediated autophagy and modulated NRF2 antioxidant response genes, as well as catalase enzymatic activity. CONCLUSIONS: C8-SA acts at two levels to protect skin against oxidative stress: 1) it prevents protein oxidation by stimulating endogenous antioxidant defense and 2) it increases the clearance of oxidized proteins by stimulating chaperone-mediated autophagy. These results suggest that C8-SA maintains skin health in urban polluted environments.

Alpha repeat proteins (αRep) as expression and crystallization helpers.

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.

Structural Analysis of the Hanks-Type Protein Kinase YabT From Bacillus subtilis Provides New Insights in its DNA-Dependent Activation.

YabT is a serine/threonine kinase of the Hanks family from Bacillus subtilis, which lacks the canonical extracellular signal receptor domain but is anchored to the membrane through a C-terminal transmembrane helix. A previous study demonstrated that a basic juxtamembrane region corresponds to a DNA-binding motif essential for the activation of YabT trans-autophosphorylation. YabT is expressed during spore development and localizes to the asymmetric septum where it specifically phosphorylates essential proteins involved in genome maintenance, such as RecA, SsbA, and YabA. YabT has also been shown to phosphorylate proteins involved in protein synthesis, such as AbrB and Ef-Tu, suggesting a possible regulatory role in the progressive metabolic quiescence of the forespore. Finally, cross phosphorylations with other protein kinases implicate YabT in the regulation of numerous other cellular processes. Using an artificial protein scaffold as crystallization helper, we determined the first crystal structure of this DNA-dependent bacterial protein kinase. This allowed us to trap the active conformation of the kinase domain of YabT. Using NMR, we showed that the basic juxtamembrane region of YabT is disordered in the absence of DNA in solution, just like it is in the crystal, and that it is stabilized upon DNA binding. In comparison with its closest structural homolog, the mycobacterial kinase PknB allowed us to discuss the dimerization mode of YabT. Together with phosphorylation assays and DNA-binding experiments, this structural analysis helped us to gain new insights into the regulatory activation mechanism of YabT.

New aminopyrimidine derivatives as inhibitors of the TAM family.

In this study, we describe the synthesis of new pyrimidine analogs of BMS-777607, a potent and selective inhibitor of Met kinase. Inhibition of Met and Axl remained high whereas inhibition of Tyro3 and Mer decreased to some extend. The preferential moderate inhibition of the non-phosphorylated form of Abl1 of some derivatives suggests that they behave as type II inhibitors. This hypothesis was confirmed by docking studies into the structure of Met (3F82) and in a Tyro3 model where key interactions with the hinge region, the DFG-out motif and the allosteric pocket explain this inhibition.

Inhibitors of the TAM subfamily of tyrosine kinases: synthesis and biological evaluation.

The TAM subfamily of Receptor Tyrosine Kinases (RTKs) contains three human proteins of therapeutical interest, Axl, Mer, and Tyro3. Our goal was to design a type II inhibitor specific for this family, i.e. able to interact with the allosteric pocket and with the hinge region of the kinase. We report the synthesis of several series of purine analogues of BMS-777607. The structural diversity of the designed inhibitors was expected to modify the interactions formed in the binding site and consequently to modulate their selectivity profiles. The most potent inhibitor 6g exhibits Kds of 39, 42, 65 and 200 nM against Axl, Mer, Met and Tyro3 respectively. Analysis of the affinity of 6g for active and inactive forms of Abl1, an RTK protein that does not belong to the TAM subfamily, together with the binding modes of 6g predicted by docking studies, indicates that 6g displays some selectivity for the TAM family and may act as a type II inhibitor.

The potent antiplasmodial calmodulin-antagonist trifluoperazine inhibits plasmodium falciparum calcium-dependent protein kinase 4.

Due to their critical involvement in the execution of the malaria parasite developmental pattern both in the mosquito vector and in the human host, Plasmodium calcium-dependent protein kinases (CDPKs) are considered promising candidates for the development of new tools to block malaria transmission. We report here that the phenothiazine trifluoperazine non-competitively inhibits Plasmodium falciparum CDPK4 in the micromolar range while other calmodulin antagonists only marginally affect the enzyme activity, and we propose the inhibition mechanism. Our results demonstrate that selective enzyme inhibition is achievable by targeting its calmodulin-like domain. This observation could be exploited for the discovery of innovative phenothiazine-based CDPK inhibitors of potential medical interest.

Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+) and Cu(2+) but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+) and Cu(2+) ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.