Comparison of the isomerization mechanisms of human melanopsin and invertebrate and vertebrate rhodopsins

Comparative modeling and ab initio multiconfigurational quantum chemistry are combined to investigate the reactivity of the human nonvisual photoreceptor melanopsin. It is found that both the thermal and photochemical isomerization of the melanopsin 11-cis retinal chromophore occur via a space-saving mechanism involving the unidirectional, counterclockwise twisting of the =C11H-C12H= moiety with respect to its Lys340-linked frame as proposed by Warshel for visual pigments [Warshel A (1976) Nature 260(5553):679–683]. A comparison with the mechanisms documented for vertebrate (bovine) and invertebrate (squid) visual photoreceptors shows that such a mechanism is not affected by the diversity of the three chromophore cavities. Despite such invariance, trajectory computations indicate that although all receptors display less than 100 fs excited state dynamics, human melanopsin decays from the excited state ∼40 fs earlier than bovine rhodopsin. Some diversity is also found in the energy barriers controlling thermal isomerization. Human melanopsin features the highest computed barrier which appears to be ∼2.5 kcal mol⁻¹ higher than that of bovine rhodopsin. When assuming the validity of both the reaction speed/quantum yield correlation discussed by Warshel, Mathies and coworkers [Weiss RM, Warshel A (1979) J Am Chem Soc 101:6131–6133; Schoenlein RW, Peteanu LA, Mathies RA, Shank CV (1991) Science 254(5030):412–415] and of a relationship between thermal isomerization rate and thermal activation of the photocycle, melanopsin turns out to be a highly sensitive pigment consistent with the low number of melanopsin-containing cells found in the retina and with the extraretina location of melanopsin in nonmammalian vertebrates.For a long time it was assumed that the human retina contains only two types of photoreceptor cells: the rods and cones responsible for dim-light and daylight vision, respectively. However, recent studies have revealed the existence of a small number of intrinsically photosensitive retinal ganglion cells (ipRGCs) that regulate nonvisual photoresponses (1). ipRGCs express an atypical opsin-like protein named melanopsin (2, 3) which plays a role in the regulation of unconscious visual reflexes and in the synchronization of endogenous physiological responses to the dawn/dusk cycle (circadian rhythms) (4, 5).Melanopsins are unique among vertebrate photoreceptors because their amino acid sequence shares greater similarity to invertebrate than vertebrate rhodopsin (i.e., the photoreceptor of rods) (6, 7). Like rhodopsins, melanopsins feature an up–down bundle architecture of seven transmembrane α-helices incorporating the 11-cis isomer of retinal as a covalently bound protonated Schiff base (PSB11 in Fig. 1A). Light-induced (i.e., photochemical) isomerization of PSB11 to its all-trans isomer (PSBAT) triggers an opsin conformational change that, ultimately, activates the receptor and signaling cascade (8, 9). However, similar to invertebrate and in contrast to vertebrate rhodopsins, melanopsins are bistable (10). Indeed, although vertebrate rhodopsins need a retinoid cycle (11) to regenerate PSB11, melanopsins have an intrinsic light-driven chromophore regeneration function via PSBAT back-isomerization. Furthermore, past studies have shown that melanopsins use an invertebrate-like signal transduction cascade (12).Open in a separate windowFig. 1.PSB11 chromophore reactivity. (A) Chromophore structure and isomerization to PSBAT. (B) Schematic representation of the photochemical (full arrows) and thermal (dashed arrows) isomerization paths. The CI is located energetically above the TS, features a different geometrical structure, and drives a far-from-equilibrium process. ΔE_S1-S0, τ_cis→trans, and Ea^T (in red) are the fundamental quantities computed in the present work.Melanopsins are held responsible for photoentrainment, using the changes of irradiance and spectral composition to adjust the circadian rhythm (13). The different studies carried out so far on melanopsin light sensitivity do not lead to consistent results. Although Do et al. (14) argue that ipRGCs work at extremely low irradiation intensities showing a single-photon response larger than rods, Ferrer et al. (15) conclude that the melanopsin has a reduced sensitivity relative to visual pigments. On the other hand, these photoreceptors would be expected to display high light sensitivity (14). In the vertebrate retina their density is 10⁴ times lower than that of rhodopsins. Moreover, the receptor is not confined in a dedicated cellular domain such as the outer segment of rods and cones, resulting in a ipRGCs photon capture more than 10⁶-fold lower than that of rods and cones per unit of retina illumination. A high sensitivity of melanopsins would also be consistent with their presence in extraretina locations such as in pineal complex, deep brain, and derma of nonmammalian vertebrates (e.g., amphibian) (16–18). The amount of light that can penetrate into such regions is limited and enriched in the red component due to light scattering by the surrounding tissues (14).The molecular-level understanding of the primary light response of melanopsin is a prerequisite for the comprehension of more complex properties such as its activation and sensitivity. Despite numerous studies carried out since its discovery (16), there is presently little information on the molecular mechanism of melanopsin activation. The common PSB11 chromophore of melanopsins and rhodopsins does not guarantee that the same mechanism operates in both photoreceptors. This not only concerns light-induced activation but also thermal activation: a process whose rate limits the photoreceptor light sensitivity and that is currently associated with thermal, rather than photochemical, PSB11 isomerization (19–24).The mechanism of light-induced PSB11 isomerization in vertebrate rhodopsins has been extensively investigated. Spectroscopic studies have shown that in bovine rhodopsin (Rh) the isomerization occurs on a subpicosecond timescale (25–27). Moreover, the observation of ground state (S₀) vibrational coherence (28) is consistent with a direct transfer of the excited state (S₁) population to the photoproduct (Fig. 1B) passing through a conical intersection (CI). Such a path has been located along the S₁ potential energy surface by constructing a multiconfigurational quantum chemistry (MCQC) based computer model of the photoreceptor (29–31) and spectroscopically supported by probing in the infrared (31). More recently (32), the same computer model has been used to map the Rh thermal isomerization path (Fig. 1B) providing information on the transition states controlling the reaction.Here we present a computational study focusing on the mechanism of photochemical and thermal isomerization of human melanopsin (hMeOp). This would require the construction of a computer model of hMeOp starting from the receptor crystal structure. However, the lack of hMeOp crystallographic data does not allow the use of the protocol previously applied in Rh studies. The significant sequence similarity between squid rhodopsin (sqRh), whose crystal structure is available (PDB code: 2Z73) (33), and hMeOp (40%, SI Appendix, Fig. S1) provides the fundamentals for constructing a structural model of hMeOp at a significant atomic resolution. Building on a study by Batista and coworkers (34) on murine melanopsin, we combine comparative modeling of hMeOp with MCQC to construct a quantum mechanics/molecular mechanics (QM/MM) computer model capable of simulating the photochemical and thermal isomerization reactions of hMeOp. The results are then compared with those found using Rh and sqRh models constructed using the same protocol. Such a comparison is expected to provide information on the differences in spectral and functional properties of these evolutionary distant pigments. As we will show below, the models indicate that hMeOp has a faster photochemical isomerization dynamics and a higher thermal isomerization barrier than both Rh and sqRh.     

Drug-induced solar urticaria due to tetracycline

Solar urticaria is an uncommon disorder characterized by pruritus, erythema and whealing commencing within minutes of exposure to ultraviolet (UV) and visible light, and generally resolves in a few hours. We describe a 28-year-old woman who developed pruritus and erythema 5 min after sun exposure while on tetracycline for treatment of perioral dermatitis. Phototesting elicited urticarial reactions in the UVA, UVB and visible spectra. Repeat phototesting after cessation of tetracycline was negative. This report documents the first case of solar urticaria induced by tetracycline.     

Pityriasis alba: a study of pathogenic factors

BACKGROUND: The aetiology of pityriasis alba (PA), a common dermatosis in childhood, is still controversial. The objective of this study was to assess the possible aetiopathogenic factors of this disease in infants. METHODS: Forty-four patients with PA and 31 healthy children were examined and compared. Personal hygiene habits, sun exposure, presence of Staphylococcus aureus in nasal fossae and presence of major or minor signs of atopy were assessed during anamnesis and physical examination. Susceptibility to ultraviolet (UV) B radiation was measured by the onset of a contact hypersensitivity reaction to diphenylcyclopropenone in individuals sensitized in previously irradiated areas. RESULTS: The prevalence of PA was higher in individuals with darker skin, in high phototype categories, as well as in males. The number of daily baths and sun exposure between 10.00 h and 15.00 h were significantly higher in the PA group when compared with controls (P = 0.03 and P = 0.0015, respectively). The presence of atopy signs was more common in pityriasis patients (P = 0.002). Susceptibility to UVB radiation was 29.6% in the PA group vs. 29.0% in the control group; nevertheless, important differences were found after stratification in order to control possible confounding factors. The presence of S. aureus in the nostrils was equal in both groups. CONCLUSIONS: Our results confirm that PA, in our population, is more prevalent in males and in individuals in higher phototype categories. In those with inadequate personal hygiene and sun exposure habits the disease is more accentuated, demonstrating that the xerosis presenting in individuals with atopic diathesis is an important element in the development of the disease. S. aureus is not an important aetiopathogenic factor in PA. Susceptibility to UVB becomes important when related to the patient's phototype.     

Threshold level for measurement of UV sensitivity: reproducibility of phototest

The ultraviolet (UV) sensitivity is determined by a phototest where the skin is exposed to well-defined doses of UV radiation and the resulting erythema is graded by visual scoring after 20–24 h. In this study we wanted to estimate the reproducibility of erythema assessment in phototesting. Twenty-one healthy Caucasians with skin types I to IV were phototested on UV un-exposed buttock skin using a xenon lamp solar simulator. Twenty-four hours after UV exposure eight physicians independently graded the erythema reactions two times. Data were analysed using inter-and intra-observer agreement and kappa statistics, which adjusts for agreement that could be caused by chance alone. Observed agreement and kappa statistics were found to decrease with increasing intensity of erythema and to be lower for skin types III and IV compared to skin types I and II. Intra-observer agreement was uniformly better than inter-observer agreement. The difference between observers assessment could be as much as three clinical erythema grades. Physicians's previous experience with phototesting only had a minor influence on agreement. In conclusion, phototesting is based on subjective assessment of erythema and is not as precise and reproducible as expected. Agreement was better for barely perceptible erythema than for erythema with a well-defined border and we therefore recommend that the barely perceptible erythema reaction should be used for measurement of the minimal erythema dose.     

The differential effect of broad band vs narrow band UVB with respect to photodamage and cutaneous inflammation

Since Fischer reported on the superiority of 313 nm UVB compared with broad band UVB in the treatment of psoriasis, narrow band UVB has become the main phototherapeutical modality in several countries. There is some discussion about the safety and photobiological effects of narrow band UVB. In the present study, narrow and broad band UVB have been compared with respect to parameters for photodamage and inflammation. Fourteen healthy volunteers were randomized in two groups. Both groups were irradiated with three minimal erythema doses (MED) of narrow or broad brand UVB, respectively. Before and 4, 24 and 48 h after irradiation, 6 mm biopsies were taken from immunohistochemical analysis of p53, apoptosis and p16 (photodamage parameters) and T-cells, polymorphonuclear leukocytes (PMN) and Langerhans' cells (inflammatory cells). Mean MED for narrow band UVB was 8.125 times higher than broad band UVB. Significant changes in expression were seen for all parameters except for p16. P53, apoptosis, T-cells and PMN increased, while Langerhans' cell count decreased significantly. No significant differences were seen between the narrow band UVB. In conclusion, following irradiation of three MED narrow band UVB and broad band UVB safety parameters for carcinogenesis and inflammation were induced to the same extent. As narrow band UVB is more effective than broad band UVB, the present study suggests superiority of narrow band UVB as a treatment with a better benefit risk ration.     

UV emissions from low energy artificial light sources

Energy efficient light sources have been introduced across Europe and many other countries world wide. The most common of these is the Compact Fluorescent Lamp (CFL), which has been shown to emit ultraviolet (UV) radiation. Light Emitting Diodes (LEDs) are an alternative technology that has minimal UV emissions. This brief review summarises the different energy efficient light sources available on the market and compares the UV levels and the subsequent effects on the skin of normal individuals and those who suffer from photodermatoses.     

Ultraviolet AI exposure of human skin results in Langerhans cell depletion and reduction of epidermal antigen-presenting cell function: partial protection by a broad-spectrum sunscreen

BACKGROUND: Ultraviolet (UV) B-induced effects on the skin immune system have been extensively investigated, but little is known regarding the immunological changes induced by UVA exposure of human skin. Recent data assessing the protection afforded by sunscreens against photoimmunosuppression stress the need for broad-spectrum sunscreens with an adequate UVA protection. OBJECTIVES: The purpose of this study was first to determine the changes observed in epidermal Langerhans cells (ELC) density and epidermal antigen-presenting cell (APC) activity after exposure of human skin to UVAI (340-400 nm) radiation, and secondly to assess the immune protection afforded in vivo by a sunscreen formulation containing a long wavelength UVA filter with a low UVA protection factor (UVA-PF = 3). METHODS: Epidermal cell (EC) suspensions were prepared from skin biopsies 3 days after exposure to a single dose of UVAI (either 30 or 60 J cm(-2)). RESULTS: Flow-cytometric analysis of EC suspensions revealed that exposure to 60 J cm(-2) UVAI resulted in a decreased number of ELC without infiltration of CD36+ DR+ CD1a- antigen-presenting macrophages into the epidermis, and a significant reduction of HLA-DR expression on viable ELC. In vivo exposure to both 30 and 60 J cm(-2) resulted in a decreased allogeneic CD4+ T-cell proliferation induced by UVAI-irradiated ECs. The sunscreen application partially prevented (57 +/- 9%) the decrease in epidermal allogeneic APC activity induced by 60 J cm(-2) UVAI. CONCLUSIONS: In vivo UVAI exposure of human skin results in a decreased number of ELC and in a downregulation of epidermal APC activity. This last effect is partially prevented by prior application of a sunscreen with a low UVAI-PF value. These results indicate that increasing the absorption of UV filters for long UVA wavelengths may lead to an improved immune protection.     

Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro

The effects of low level laser (LLL) irradiation on the proliferation of human buccal fibroblasts were studied. A standardized LLL set-up was developed (812 nm, 4.5 ± 0.5 mW/cm2). Cultures in petridishes were divided into eight groups (1 group served as control). On day 6 after seeding, routine growth medium was replaced with PBS for 1/2 hour. At the beginning of this period, LLL irradiation was performed for 0, 1, 3, 10, 32, 100, 316, or 1,000 seconds, respectively—corresponding to the radiant exposures 0, 4.5, 13.5, 45, 144, 450, 1,422, 4,500 mJ/cm2. Subsequently the cells received 3H-dT in fresh medium for 16 hours DNA-incorporation. Scintillations from tritium and total protein concentration per culture dish were determined. The individual 3H-cpm/protein-concentration ratios were calculated in % of control. Three experiments were performed (N = 151). Following LLL exposure the H-cpm/protein ratio was increased with maximum cpm/protein ratio (132.5% ± 10.6% SEM) in the group receiving 450 mJ/cm2 (P < 0.03 nonparametric Kruskal Wallis one-way ANOVA-test). This study demonstrated an increased incorporation on tritiated thymidine in cultured human oral fibroblasts following LLL exposure and suggests that LLL irradiation can induce increased DNA Synthesis. © 1994 Wiley-Liss, Inc.