The process for the activation of frog epidermis pro-tyrosinase.
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ABSTRACT:
SUBMITTER: Penafiel R
PROVIDER: S-EPMC1158493 | biostudies-other | 1982 Aug
REPOSITORIES: biostudies-other
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Project description:1. Frog epidermis tyrosinase was coupled to Sepharose activated with low concentrations of CNBr. The tetrameric form of the enzyme was linked to the matrix via its subunits. Dissociation of the bound active enzyme with guanidinium chloride yielded an active immobilized dimeric derivative. 2. Immobilized dimeric derivative was able to interact with soluble subunits formed transiently during renaturation. An 85% recovery of the native dopa oxidase specific activity was achieved after hybridization. 3. Fluorescence spectra of different immobilized derivatives suggested that tryptophan residues were involved in the interactions between tyrosinase subunits. 4. It is suggested that the activation of the subunits of tyrosinase involves a conformational change towards a more unfolded state, which favours a reassociation to the dimeric active state.
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Project description:Adenosine 5'-triphosphate (ATP) is an important extracellular signaling agent, operating in growth regulation, stomatal conductance, and wound response. With the first receptor for extracellular ATP now identified in plants (P2K1/DORN1) and a plasma membrane NADPH oxidase revealed as its target, the search continues for the components of the signaling cascades they command. The Arabidopsis root elongation zone epidermal plasma membrane has recently been shown to contain cation transport pathways (channel conductances) that operate downstream of P2K1 and could contribute to extracellular ATP (eATP) signaling. Here, patch clamp electrophysiology has been used to delineate two further conductances from the root elongation zone epidermal plasma membrane that respond to eATP, including one that would permit chloride transport. This perspective addresses how these conductances compare to those previously characterized in roots and how they might operate together to enable early events in eATP signaling, including elevation of cytosolic-free calcium as a second messenger. The role of the reactive oxygen species (ROS) that could arise from eATP's activation of NADPH oxidases is considered in a qualitative model that also considers the regulation of plasma membrane potential by the concerted action of the various cation and anion conductances. The molecular identities of the channel conductances in eATP signaling remain enigmatic but may yet be found in the multigene families of glutamate receptor-like channels, cyclic nucleotide-gated channels, annexins, and aluminum-activated malate transporters.
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Project description:Specialised epithelia such as mucociliary, secretory and transporting epithelia line all major organs, including the lung, gut and kidney. Malfunction of these epithelia is associated with many human diseases. The frog embryonic epidermis possesses mucus-secreting and multiciliated cells, and has served as an excellent model system for the biogenesis of cilia. However, ionic regulation is important for the function of all specialised epithelia and it is not clear how this is achieved in the embryonic frog epidermis. Here, we show that a third cell type develops alongside ciliated and mucus-secreting cells in the tadpole skin. These cells express high levels of ion channels and transporters; therefore, we suggest that they are analogous to ionocytes found in transporting epithelia such as the mammalian kidney. We show that frog ionocytes express the transcription factor foxi1e, which is required for the development of these cells. Depletion of ionocytes by foxi1e knockdown has detrimental effects on the development of multiciliated cells, which show fewer and aberrantly beating cilia. These results reveal a newly identified role for ionocytes and suggest that the frog embryonic skin is a model system that is particularly suited to studying the interactions of different cell types in mucociliary, as well as in secretory and transporting, epithelia.
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Project description:The conversion of inactive pro-polyphenol oxidases (pro-PPOs) into the active enzyme results from the proteolytic cleavage of its C-terminal domain. Herein, a peptide-mediated cleavage process that activates pro-MdPPO1 (Malus domestica) is reported. Mass spectrometry, mutagenesis studies, and X-ray crystal-structure analysis of pro-MdPPO1 (1.35 Å) and two separated C-terminal domains, one obtained upon self-cleavage of pro-MdPPO1 and the other one produced independently, were applied to study the observed self-cleavage. The sequence Lys 355-Val 370 located in the linker between the active and the C-terminal domain is indispensable for the self-cleavage. Partial introduction (Lys 352-Ala 360) of this peptide into the sequence of two other PPOs, MdPPO2 and aurone synthase (CgAUS1), triggered self-cleavage in the resulting mutants. This is the first experimental proof of a self-cleavage-inducing peptide in PPOs, unveiling a new mode of activation for this enzyme class that is independent of any external protease.
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Project description:Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NF?B, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions.
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Project description:We confirmed Tyrosinase genome insertions or deletions of the three Tg mice by deep sequencing using MiSeq. These mice generated gene editing mice by using Tol2 transposon to introduce tyrosinase guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-Cas9 mice with CAG-CreER mice.
2023-02-27 | GSE225711 | GEO
Project description:beta-Catenin signaling is required for hair follicle development, but it is unknown whether its activation is sufficient to globally program embryonic epidermis to hair follicle fate. To address this, we mutated endogenous epithelial beta-catenin to a dominant-active form in vivo. Hair follicle placodes were expanded and induced prematurely in activated beta-catenin mutant embryos, but failed to invaginate or form multilayered structures. Eventually, the entire epidermis adopted hair follicle fate, broadly expressing hair shaft keratins in place of epidermal stratification proteins. Mutant embryonic skin was precociously innervated, and displayed prenatal pigmentation, a phenomenon never observed in wild-type controls. Thus, beta-catenin signaling programs the epidermis towards placode and hair shaft fate at the expense of epidermal differentiation, and activates signals directing pigmentation and innervation. In transcript profiling experiments, we identified elevated expression of Sp5, a direct beta-catenin target and transcriptional repressor. We show that Sp5 normally localizes to hair follicle placodes and can suppress epidermal differentiation gene expression. We identified the pigmentation regulators Foxn1, Adamts20 and Kitl, and the neural guidance genes Sema4c, Sema3c, Unc5b and Unc5c, as potential mediators of the effects of beta-catenin signaling on pigmentation and innervation. Our data provide evidence for a new paradigm in which, in addition to promoting hair follicle placode and hair shaft fate, beta-catenin signaling actively suppresses epidermal differentiation and directs pigmentation and nerve fiber growth. Controlled downregulation of beta-catenin signaling is required for normal placode patterning within embryonic ectoderm, hair follicle downgrowth, and adoption of the full range of follicular fates.
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Project description:Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 ? resolution, respectively, revealing that Phe(513) on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193-201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19-26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys(92), which is covalently bound to His(94) via an unusual thioether linkage in the holo-form, and Cys(522) and Cys(525) of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.
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