Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Trans-urocanic acid facilitates spatial memory, implications for Alzheimer's disease.

Trans-urocanic acid (trans-UCA) is an isomer of cis-UCA and is widely distributed in the brain, predominantly in the hippocampus and prefrontal cortex. Previous studies have investigated the role of trans-UCA in non-spatial memory; however, its influence on spatial memory remains unclear. In the present study, network pharmacology strategy and behavioral testing were used to evaluate the role of trans-UCA in spatial memory and predict its possible mechanism. The results showed that there are 40 intersecting targets between trans-UCA and spatial memory identified by several databases and Venn diagram, indicating that trans-UCA may be involved in spatial memory. Behavioral results show that trans-UCA facilitates spatial working memory in the Y-maze test as well as spatial recognition memory acquisition, consolidation and retrieval in an object location recognition (OLR) task. Furthermore, PPI (protein-protein interaction) network analysis, GO (gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analyses show that the molecular mechanisms underlying the enhancing effect of trans-UCA on spatial memory are mainly associated with the regulation of insulin, mitogen-activated protein kinase (MAPK) and nuclear factor Kappa B (NF-κB) signaling pathways, serotonergic synapse and arginine and proline metabolism. The results of this study suggest that trans-UCA facilitates spatial memory in the Y-maze test and OLR task and may offer therapeutic potential for Alzheimer's disease (AD). The underlying mechanisms predicted by network pharmacology should be further verified.

The dark side of daylight: photoaging and the tumor microenvironment in melanoma progression.

Continued thinning of the atmospheric ozone, which protects the earth from damaging ultraviolet radiation (UVR), will result in elevated levels of UVR reaching the earth's surface, leading to a drastic increase in the incidence of skin cancer. In addition to promoting carcinogenesis in skin cells, UVR is a potent extrinsic driver of age-related changes in the skin known as "photoaging." We are in the preliminary stages of understanding of the role of intrinsic aging in melanoma, and the tumor-permissive effects of photoaging on the skin microenvironment remain largely unexplored. In this Review, we provide an overview of the impact of UVR on the skin microenvironment, addressing changes that converge or diverge with those observed in intrinsic aging. Intrinsic and extrinsic aging promote phenotypic changes to skin cell populations that alter fundamental processes such as melanogenesis, extracellular matrix deposition, inflammation, and immune response. Given the relevance of these processes in cancer, we discuss how photoaging might render the skin microenvironment permissive to melanoma progression.

Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production.

The human microbiome can produce metabolites that modulate insulin signaling. Type 2 diabetes patients have increased circulating concentrations of the microbially produced histidine metabolite, imidazole propionate (ImP) and administration of ImP in mice resulted in impaired glucose tolerance. Interestingly, the fecal microbiota of the patients had increased capacity to produce ImP, which is mediated by the bacterial enzyme urocanate reductase (UrdA). Here, we describe the X-ray structures of the ligand-binding domains of UrdA in four different states, representing the structural transitions along the catalytic reaction pathway of this unexplored enzyme linked to disease in humans. The structures in combination with functional data provide key insights into the mechanism of action of UrdA that open new possibilities for drug development strategies targeting type 2 diabetes.

The Multiple Roles of Urocanic Acid in Health and Disease.

Trans-urocanic acid (trans-UCA) is synthesized in the skin, liver, and brain. It is a major natural moisturizing factor in skin and maintains its acid pH. In skin, it isomerizes to cis-UCA following exposure to UVR. Both isomers fulfill multiple roles in health and disease. Cis-UCA has immunomodulatory properties linked with several cutaneous diseases such as skin cancer, atopic dermatitis, and urticaria and associates with systemic diseases including multiple sclerosis. The levels of UCA in the skin, brain, urine, and feces reflect some physiological processes and may be disease biomarkers. Both isomers of UCA have therapeutic potential for a range of disorders.

Actinic keratosis and surrounding skin exhibit changes in corneocyte surface topography and decreased levels of filaggrin degradation products.

Actinic keratosis (AK) is a frequent premalignant skin lesion mainly caused by chronic sun exposure. AK lesions are often surrounded by invisible, subclinical alterations, called field of cancerization (FoC). Definition of FoC is of importance for therapy management; however, the criteria and non-invasive tools to characterize FoC are lacking. Atomic force microscopy (AFM) proved to be a suitable tool for detection of changes in the corneocyte surface topography in inflammatory skin diseases, which share similar clinical features with AK such as hyper- and parakeratosis. Therefore, in this study we applied AFM to investigate AK and surrounding skin obtained by non-invasive collection of the stratum corneum (SC) with adhesive tapes. Furthermore, we determined degradation products of structural protein filaggrin (natural moisturizing factor, NMF), which previously showed association with the changes in corneocyte surface topography. Ten patients with multiple AK on the face were recruited from the outpatient clinic. SC samples were collected from the AK lesion, skin sites adjacent to the AK, 5 cm from the AK and retroauricular area. Corneocyte surface topography was determined by AFM, and NMF by liquid chromatography. The AK lesion showed alterations of the corneocyte surface topography characterized by an increased number of nanosize protrusions, which gradually decreased with the distance from the lesion. NMF levels show an inverse pattern. Atomic force microscopy showed to be a suitable tool to detect changes in the corneocyte surface topography on the AK lesion and surrounding skin in a non-invasive manner.

The Influence of Calcium on the Skin pH and Epidermal Barrier During Aging.

During aging, the pH of the epidermis goes up and the calcium gradient goes down. Both have negative effects on the protective function of the epidermis and both are connected to each other as is discussed here. In the aging process, the pH rises from ∼5 to ∼5.5-6. The establishment of the skin pH is the joint effort of several independent factors including the activity of sodium-hydrogen antiporters and the presence of lactate, urocanic acid, free fatty acids and melanophores in the outermost layers of the skin. All these elements are under the control of a small ion: calcium. Calcium is organized in the form of a gradient in the epidermis with low concentrations in the stratum basale and peak concentrations in the stratum granulosum. During the aging process, this epidermal gradient collapses. In this chapter, we describe how a drop of calcium in the stratum granulosum affects the expression as well as the activity of proteins and enzymes that are involved in the establishment of the skin pH. This rise of the pH combined with a rearrangement of the cornified envelope is a main driver for a reduced epidermal barrier in old age leading to an increased prevalence for infections, reduced resistance against mechanical stress and reduced wound healing.

Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain.

Sunlight exposure is known to affect mood, learning, and cognition. However, the molecular and cellular mechanisms remain elusive. Here, we show that moderate UV exposure elevated blood urocanic acid (UCA), which then crossed the blood-brain barrier. Single-cell mass spectrometry and isotopic labeling revealed a novel intra-neuronal metabolic pathway converting UCA to glutamate (GLU) after UV exposure. This UV-triggered GLU synthesis promoted its packaging into synaptic vesicles and its release at glutamatergic terminals in the motor cortex and hippocampus. Related behaviors, like rotarod learning and object recognition memory, were enhanced after UV exposure. All UV-induced metabolic, electrophysiological, and behavioral effects could be reproduced by the intravenous injection of UCA and diminished by the application of inhibitor or short hairpin RNA (shRNA) against urocanase, an enzyme critical for the conversion of UCA to GLU. These findings reveal a new GLU biosynthetic pathway, which could contribute to some of the sunlight-induced neurobehavioral changes.

[The epidermal barrier]. / Barrière épidermique.

The skin acts as an interface between the body and its surrounding environment. The epidermis, the surface layer of the skin, is chiefly responsible for this interactive protective function. The epidermal barrier may be subdivided into three defensive systems: the photoprotective barrier, the immune barrier, and the physical and chemical barrier of the stratum corneum or horny layer. To protect against harmful ultraviolet radiation, the epidermis has absorption factors such as melanin, produced by melanocytes, and urocanic acid, which is a degradation product of filaggrin. The epidermal immune defence system comprises an innate component, which is rapid but non-specific, together with adaptive response, which is systemic and antigen-specific, initiated by Langerhans cells. The stratum corneum, derived from terminal differentiation of epidermal keratinocytes, plays a key role as a physical and chemical permeability barrier. This horny layer is made up of corneocytes, covered with horny envelopes and linked to one another by corneodesmosomes and by extracellular matrix sheets. The epidermal barrier, which is constantly being renewed, is characterised by its extremely great capacity of adaptation to changing conditions in the environment.

Loss-of-function mutations in filaggrin gene and malignant melanoma: a case-control study.

BACKGROUND: Loss-of-function mutations in filaggrin gene (FLG) have been suggested to increase the susceptibility of skin malignancies due to reduced levels of epidermal filaggrin and its degradation products, urocanic acid, which may be protective against ultraviolet irradiation. OBJECTIVE: We aimed to investigate the association between FLG mutation status and the occurrence of malignant melanoma (MM) in Danish adults. METHODS: The prevalence of FLG mutations in a sample of MM biopsies was compared with a FLG-genotyped cohort from two general population studies. Pearson's chi-squared and Fisher's exact tests were used to compare the two groups. RESULTS: A total of 867 MM biopsies and 9965 general population controls were genotyped, respectively. In the MM sample, two (0.23%) individuals were homozygous and 80 (9.4%) were heterozygous mutation carriers. In the general population controls, the prevalence of FLG mutations was 18 (0.18%) and 835 (8.4%) for homozygous and heterozygous mutations, respectively. Fisher's exact test and Pearson's chi-squared test yielded non-significant P-values when the groups were compared. CONCLUSION: FLG mutation was not associated with MM in the studied populations. This finding indicates that epidermal deficiency of filaggrin and its degradation products does not influence the risk of MM significantly.

Increased cis-to-trans urocanic acid ratio in the skin of chronic spontaneous urticaria patients.

Increased filaggrin expression was found to be correlated with severity scores in chronic spontaneous urticaria (CSU); however, the role of filaggrin breakdown products (FBPs) in CSU has not been studied. We collected stratum corneum (SC) specimens from the volar forearms of 10 CSU patients, 10 AD patients, and 10 healthy normal controls (NCs) and measured contents of FBPs (pyrrolidone carboxylic acid [PCA] and urocanic acid [UCA]) using UPLC-MS/MS, transepidermal water loss (TEWL) and epidermal pH. Compared to NCs, cis-UCA level was increased in CSU lesions (P < 0.05) and decreased in AD lesions (P < 0.01). The cis-to-trans-UCA ratio in SC specimens from CSU patients was significantly greater than those from AD and NC subjects. AD lesions had lower FBP and PCA contents compared to NC skin (both P < 0.001), and higher TEWL and pH compared to CSU lesions. Moreover, cis-UCA, but not trans-UCA, enhanced the IgE-mediated basophil activation, as well as IgE- and calcium-mediated degranulation of LAD-2 cells, in a dose-dependent manner. These findings suggest that increased cis-to-trans UCA ratio in the epidermis is a distinct feature of CSU, which could enhance mast cell degranulation. Modulation of cis-UCA may be a potential target for skin diseases associated with IgE-mediated mast cell degranulation.

Expression of Filaggrin and its Degradation Products in Human Skin Following Erythemal Doses of Ultraviolet B Irradiation.

Epidermal filaggrin level is affected by ultraviolet B irradiation in animal and experimental models. This study examined the effect of ultraviolet B irradiation on epidermal filaggrin and natural moisturizing factors in vivo in healthy adults (n = 22). Participants were irradiated with 2 minimal erythema doses of ultraviolet B on the skin. Biopsies and tape strips were collected from skin irradiated 24 and 72 h earlier and from non-irradiated skin (control). Real-time quantitative PCR on skin biopsies showed significantly reduced profilaggrin mRNA expression 24 h after irradiation (mean relative mRNA expression ± standard deviation: control, 3.86 ± 2.06 vs. 24 h, 1.52 ± 0.640; p = 0.02; n = 8). Immunohistochemistry showed aberrant spatial distribution of filaggrin protein 72 h after irradiation (n = 3). High-pressure liquid chromatography of tape extracts showed no change in mean total natural moisturizing factor levels after irradiation, but mean trans-urocanic acid was significantly reduced, as expected (n = 8). In conclusion, erythemal doses of ultraviolet B exert acute effects on profilaggrin mRNA and filaggrin protein in human skin in vivo.

The effect of epidermal levels of urocanic acid on 25-hydroxyvitamin D synthesis and inflammatory mediators upon narrowband UVB irradiation.

BACKGROUND/PURPOSE: Urocanic acid (UCA) absorbs ultraviolet (UV)B radiation in the epidermis which may interfere with phototherapy. Therefore, the influence of individual levels of UCA on immune reactivity and vitamin D synthesis induced by narrowband UVB radiation was assessed. METHODS: Twenty-eight subjects with irritant contact dermatitis of the hands were irradiated with suberythemal doses of narrowband UVB radiation on their unaffected lower forearms on three consecutive days. Stratum corneum tape strips and epidermal interstitial fluid (ISF) as well as blood samples were analyzed. RESULTS: Narrowband UVB irradiation led to the conversion of trans-UCA into its cis-isomer in the epidermis. The observed increase in 25-hydroxyvitamin D serum concentrations was inversely correlated with the baseline levels of trans-UCA. Furthermore, UVB irradiation induced significant changes in the levels of CXCL10/IP-10, CCL2/MCP-1, CCL4/MIP-1ß, and the IL-1RA/IL-1α ratio. The levels of IL-1α and CXCL9/MIG showed a trend toward increase. The changes in the levels of inflammatory and immunomodulatory mediators did not depend on baseline levels of trans-UCA. CONCLUSION: The results suggest that epidermal levels of trans-UCA affect vitamin D synthesis, but not cutaneous immune reactivity upon repeated exposure to suberythemal doses of narrowband UVB radiation. However, this requires further exploration.

Current knowledge in Polypodium leucotomos effect on skin protection.

This article provides an overview of pharmacology, toxicity, pharmacokinetics and clinical data of Polypodium leucotomos L. (PL). PL aerial part has proven to exert antioxidant, photoprotective and immunomodulatory activities; its mechanism of action is complex and includes several activities: (1) PL diminishes the production of reactive oxygen and nitrogen species (ROS, RNS); (2) PL inhibits the photoisomerization of trans-urocanic acid (t-UCA); (3) PL inhibits apoptosis induced by ultraviolet radiation; (4) PL prevents damage to genetic material and (5) PL enhances DNA repair. PL is not mutagenic and does not induce acute or chronic toxicity. Its biological effects have been proved in cell cultures, animal models, murine models and in human beings. Photoprotective activity has been assessed in healthy volunteers as well as in patients suffering from several cutaneous diseases such as vitiligo, psoriasis, idiopathic photodermatosis or melasma. PL results to be an efficient treatment especially for sensitive cutaneous phototypes and adds extra protection when ultraviolet radiation (UVR) exposure cannot be avoided, such as wide or narrow band UVB phototherapy or treatment with psoralens plus UVA exposure radiation.

Phototherapy and photochemotherapy for psoriasis.

Phototherapy is a first-line option for the treatment of moderate to severe psoriasis. Systematic reviews indicate near comparable efficacy of the different forms of phototherapy. Localized phototherapy can be an adjunctive treatment of recalcitrant plaques during systemic treatment of psoriasis. More than 200 psoralen-UV-A therapy treatment sessions is associated with an increased risk of keratinocytic cancers, whereas no increased risk has been demonstrated for narrow-band UV-B therapy. The mechanism of action of phototherapy in psoriasis is via inhibition of keratinocyte proliferation; induction of apoptosis in keratinocytes, dendritic, and T cells; and inhibition of Th1 and Th17 pathways, but activation of Th2.

The skin microbiome of caspase-14-deficient mice shows mild dysbiosis.

Caspase-14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase-14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2-pyrrolidone-5-carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase-14(-/-) mice show enhanced antibacterial response compared to wild-type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild-type and caspase-14(-/-) mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase-14 ablation leads to an increase in bacterial richness and diversity during steady-state conditions. Although both wild-type and caspase-14(-/-) skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase-14(-/-) mice. Altogether, our data demonstrated that caspase-14 deficiency causes the imbalance of the skin-resident bacterial communities.

Filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3D skin construct.

Mutations in the filaggrin (FLG) gene are strongly associated with common dermatological disorders such as atopic dermatitis. However, the exact underlying pathomechanism is still ambiguous. Here, we investigated the impact of FLG on skin lipid composition, organization, and skin acidification using a FLG knockdown (FLG-) skin construct. Initially, sodium/hydrogen antiporter (NHE-1) activity was sufficient to maintain the acidic pH (5.5) of the reconstructed skin. At day 7, the FLG degradation products urocanic (UCA) and pyrrolidone-5-carboxylic acid (PCA) were significantly decreased in FLG- constructs, but the skin surface pH was still physiological owing to an upregulation of NHE-1. At day 14, secretory phospholipase A2 (sPLA2) IIA, which converts phospholipids to fatty acids, was significantly more activated in FLG- than in FLG+. Although NHE-1 and sPLA2 were able to compensate the FLG deficiency, maintain the skin surface pH, and ensured ceramide processing (no differences detected), an accumulation of free fatty acids (2-fold increase) led to less ordered intercellular lipid lamellae and higher permeability of the FLG- constructs. The interplay of the UCA/PCA and the sPLA2/NHE-1 acidification pathways of the skin and the impact of FLG insufficiency on skin lipid composition and organization in reconstructed skin are described.

Urocanate as a potential signaling molecule for bacterial recognition of eukaryotic hosts.

Host recognition is the crucial first step in infectious disease pathogenesis. Recognition allows pathogenic bacteria to identify suitable niches and deploy appropriate phenotypes for successful colonization and immune evasion. However, the mechanisms underlying host recognition remain largely unknown. Mounting evidence suggests that urocanate-an intermediate of the histidine degradation pathway-accumulates in tissues, such as skin, and acts as a molecule that promotes bacterial infection via molecular interaction with the bacterial regulatory protein HutC. In Gram-negative bacteria, HutC has long been known as a transcriptional repressor of hut genes for the utilization of histidine (and urocanate) as sources of carbon and nitrogen. Recent work on the opportunistic human pathogen Pseudomonas aeruginosa and zoonotic pathogen Brucella abortus shows that urocanate, in conjunction with HutC, plays a significant role in the global control of cellular metabolism, cell motility, and expression of virulence factors. We suggest that in addition to being a valuable source of carbon and nitrogen, urocanate may be central to the elicitation of bacterial pathogenesis.