Female C57BL/6 (H-2b) mice, 6–10 wk old, obtained from Frederick Cancer Research Center (Frederick, MD) and maintained in a barrier facility, were used for all experiments. EL-4 thymoma (H-2b) and the derived β-galactosidase (β-gal)–transfected clone E22 have been described previously (12 ). B16 (H-2b), hereafter named B16.WT, is a spontaneous murine melanoma expressing gp100, MART-1, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 by FACS® and Western blot analysis (data not shown). B16.B7-1 is a hyperpigmented clone of B16.WT that was stably transfected using a Moloney mouse leukemia virus encoding the gene for B7-1 driven by a LTR promoter. JB/MS is a pigmented, chemically induced melanoma expressing gp100, provided by Dr. Vincent Hearing (National Cancer Institute, NIH, Bethesda, MD). 293Kb and 293KbDb are highly transfectable human renal 293 cells stably transfected with Kb and Kb plus Db, respectively. RMA/S (H-2b) is a cell line deficient in endogenous peptide loading (13 (link)). EL-4, B16.WT, RMA/S, MCA205, and MC38, a C57BL/6-derived colon carcinoma, were maintained in complete medium (CM; RPMI 1640 with 10% heat-inactivated fetal bovine serum [FBS; Biofluids, Rockville, MD], 0.03% l -glutamine, 100 μg/ml streptomycin, 100 μg/ml penicillin, and 50 μg/ml gentamycin sulfate [NIH Media Center, Bethesda, MD]). B16.B7-1 and E22 were maintained in CM with 400 μg/ml of bioactive G418. 293Kb and 293KbDb were maintained in DMEM with 10% heat-inactivated FBS, 0.03% l -glutamine, 100 μg/ml streptomycin, 100 μg/ml penicillin, 50 μg/ml gentamycin sulfate, and 400 μg/ml of bioactive G418.
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Tyrosinase-related protein-1
Tyrosinase-related protein-1
Tyrosinase-Related Protein-1 (TYRP1) is a key enzyme involved in melanin biosynthesis, playing a crucial role in pigmentation and skin coloration.
This protein is a member of the tyrosinase-related protein family and is essential for the proper synthesis and distribution of melanin.
TYRP1 research is crucial for understanding melanogenesis, pigmentation disorders, and related dermatological conditions.
PubCompare.ai, an AI-powered platform, can streamline your TYRP1 research by enhancing reproducibility and accuracy.
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This protein is a member of the tyrosinase-related protein family and is essential for the proper synthesis and distribution of melanin.
TYRP1 research is crucial for understanding melanogenesis, pigmentation disorders, and related dermatological conditions.
PubCompare.ai, an AI-powered platform, can streamline your TYRP1 research by enhancing reproducibility and accuracy.
Discover the best protocols and products by accessing literature, pre-prints, and patents, with AI-driven comparisons to identify the most reliable and effective approaches.
Optimize your research workflow and achieve better results with PubCompare.ai.
Most cited protocols related to «Tyrosinase-related protein-1»
antibiotic G 418
beta-Galactosidase
Cancer of Colon
Cell Lines
Cells
Clone Cells
dopachrome isomerase
Females
Genes
Glutamine
Homo sapiens
Kidney
Malignant Neoplasms
MART-1 Antigen
Melanoma
Monophenol Monooxygenase
Murine Leukemia Virus
Mus
Penicillins
Peptides
Streptomycin
Sulfate, Gentamicin
Thymoma
tyrosinase-related protein-1
Western Blot
HEK293T (American Type Culture Collection, ATCC; CRL-3216), FeFAB [40 (link)], CrFK (ATCC, CCL-944), and KE-R (Depository for Cell Lines in Veterinary Medicine, Federal Research Institute for Viral Animal Diseases, Riems, Germany) were propagated under standard conditions in DMEM containing 10% heat-inactivated fetal calf serum plus antibiotics [40 (link)]. CrFK-derived FeFAB (FFV-activated β-galactosidase) cells contain the β-galactosidase gene under the control of the FFV internal promoter [40 (link)]. All cells were regularly screened for the absence of mycoplasma and other agents and for cell authenticity (Multiplexion, Immenstaad, Germany).
The C57BL/6-derived tumor cell lines EL4 [41 (link)] and RMA [42 (link)] were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, and 1% penicillin-streptomycin. The transfectants E.G7 (EL4 cells expressing chicken ovalbumin) [43 (link)], 2F11 (RMA cells expressing the HPV16-derived oncoprotein E7) [44 (link)] and RMA/TRP-2 (expressing human tyrosinase related protein 2) (kindly provided by A. Paschen), were grown in the same medium containing 0.8 μg/ml G418 (Gibco). The following antigen-specific CTL lines were used: OVA-specific CTL line [45 (link)] recognizing the H2Kb-restricted OVA-derived epitope aa257-264 (SIINFEKL) [46 (link)]; E7-specific CTL line [47 (link)], reacting against the E7-derived Db-restricted epitope RAHYNIVTF (amino acids, aa, 49–57 [48 (link)], and a TRP-2-specific CTL line obtained upon TRP-2-specfic DNA immunization of C57BL/6 mice (Osen, unpublished), recognizing the epitope SVYDFFVWL (aa 180–188) [49 (link)]. All CTL lines were expanded in vitro upon periodical re-stimulation with the transfectant clones expressing the respective target antigen in CTL medium, according to the protocol described in [50 (link)].
The C57BL/6-derived tumor cell lines EL4 [41 (link)] and RMA [42 (link)] were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, and 1% penicillin-streptomycin. The transfectants E.G7 (EL4 cells expressing chicken ovalbumin) [43 (link)], 2F11 (RMA cells expressing the HPV16-derived oncoprotein E7) [44 (link)] and RMA/TRP-2 (expressing human tyrosinase related protein 2) (kindly provided by A. Paschen), were grown in the same medium containing 0.8 μg/ml G418 (Gibco). The following antigen-specific CTL lines were used: OVA-specific CTL line [45 (link)] recognizing the H2Kb-restricted OVA-derived epitope aa257-264 (SIINFEKL) [46 (link)]; E7-specific CTL line [47 (link)], reacting against the E7-derived Db-restricted epitope RAHYNIVTF (amino acids, aa, 49–57 [48 (link)], and a TRP-2-specific CTL line obtained upon TRP-2-specfic DNA immunization of C57BL/6 mice (Osen, unpublished), recognizing the epitope SVYDFFVWL (aa 180–188) [49 (link)]. All CTL lines were expanded in vitro upon periodical re-stimulation with the transfectant clones expressing the respective target antigen in CTL medium, according to the protocol described in [50 (link)].
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Chemical structures were prepared and converted in .mol files using ChemSketch software. A compound-target analysis was also conducted through the bioinformatics platform STITCH (http://stitch.embl.de/cgi/network.pl ), with the aim to predict microbial targets. Regarding the docking analysis, the routine steps for the docking calculations involved the preparation of the inhibitors and the protein. The crystal structures of the proteins were downloaded from the Protein Data Bank (PDB: https://www.rcsb.org/ ). The PDB codes were: 5I3B (tyrosinase from Bacillus megaterium), 5M8P (Human Tyrosinase Related Protein 1), 3MKT [multidrug efflux system transporters of E. coli (mdtK)], 5Y50 [MATE transporter AtDTX14], 3ESH [S. aureus β-lactamase (ORF259)]. In order to prepare the protein for docking calculations, all water molecules and co-crystalized compounds were removed. This step was followed by adding polar hydrogen atoms and neutralization using Autodock4 program (Molinspiration Database). The starting structures of secondary metabolites were optimized to their ground state structures using the AM1 semiempirical method, and the 3D structures were saved in mol2 format. The protein was immersed in a 3D grid box with 60 × 60 × 60 dimensions with 0.375 Å distance between points. The Lamarckian genetic algorithm was used to calculate the docking free energy of 250 conformations for each inhibitor. The docking results were clustered and organized according to the docking free energy. The binding site was localized, and the non-bonding interactions were elucidated using the Discovery Studio 5.0 visualizer.
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Animals
Cadherins
Dysbindin
Genetic Background
Genotype
Glycoproteins
Hemolytic Complement
Heterozygote
Homozygote
Hydrochloride, Dopamine
Locomotion
Long-Term Potentiation
Males
Memory
Mice, Inbred DBA
Mice, Laboratory
Mutation
Phenotype
Prosencephalon
Strains
Tail
TYRP1 protein, human
Biological Assay
CD4 Positive T Lymphocytes
Mus
Tail
tyrosinase-related protein-1
Veins
Most recents protocols related to «Tyrosinase-related protein-1»
Cells were cultured in a T75 flask and when the confluence reached 75%, the proteins were isolated and analyzed by mass spectrometry, as described.31 (link) Then, the raw data were analyzed by proteome discoverer version 2.2 (Thermo Electron). The presence of tyrosinase and other melanocyte differentiation antigens (microphthalmia-associated transcription factor [MITF], tyrosinase-related protein 1 [TYRP-1], tyrosinase-related protein 2 [TYRP-2], glycoprotein 100 [gp-100], and Melan-A) was determined by proteomics analysis.32 (link)
The target protein structures were obtained from http://www.rcsb.org/pdb/home/home.do and selected based on their structures in the active form bound to the native ligand. The protein targets used tyrosinase (PDB ID: 2Y9X), tyrosinase-related protein 1 (PDB ID: 5M8M), and D-dopachrome tautomerase (PDB ID: 3KAN). The preparation of the target protein was conducted using the Chimera 1.11.1 program.
The analysis of the data employed descriptive methods. Findings obtained from molecular docking included information about binding energy and the type of bond formed between the compound (luteolin) and the protein target (tyrosinase, tyrosinase-related protein 1, and D-dopachrome tautomerase). The energy value indicates the affinity between the compound and the protein target. A more negative energy value signifies a stronger binding affinity of the ligand to the protein target.
Patchouli essential oil (PEO) was provided by Bio-Jourdeness International Groups Co., Ltd. (Taichung, Taiwan). Patchouli alcohol was obtained from Biosynth International, Inc. (San Diego, CA, USA). The compound's purity was determined to be above 99%, as confirmed by both gas chromatography (GC) and proton nuclear magnetic resonance ( 1 H-NMR) analyses. Fetal bovine serum (FBS), Roswell park memorial institute (RPMI) 1640 medium, penicillin, and streptomycin were procured from Life Technologies (Grand Island, NY, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), tyrosinase (EC 1.14.18.1, activity of 6680 units/mg), melanin, and kojic acid (KA) were purchased from Sigma-Aldrich (St. Louis, CA, USA). Forskolin (FRK) was acquired from Selleckchem (Houston, TX, USA). An antibody against tyrosinase was obtained from Genetex, Irvin, CA, USA. Antibodies against GAPDH, tyrosinase-related protein-1, and tyrosinase-related protein-2 were obtained from Santa-Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase (HRP)-linked anti-mouse IgG and anti-rabbit IgG antibodies were sourced from Cell Signaling Technology (Danvers, MA, USA). All other chemicals used were of reagent grade or HPLC grade and were provided by either Merck (Darmstadt, Germany) or Sigma-Aldrich.
The protein structure of the target enzyme human Tyrosinase (hTYR) has no resolved X-ray crystallographic or cryo-EM structure available in the protein databank yet. Therefore, the amino acid sequence of this enzyme was taken from the UniProt database with sequence ID-P14679 and a new protein structure of this enzyme was homology modelled by utilizing the SWISS-MODEL51 (link) server. The template (used) for modelling this structure, was hTYRP1 (human tyrosinase-related protein-1) with PDB ID-5M8L 52 as this enzyme showed the highest homology with the human tyrosinase. As the hTYRP1 contains the Zn++ as active catalytic atoms and the hTYR contains the Cu++ ions utilizing the MOE v2022 software, the copper ions were modelled into the prepared homology modelled hTYR enzyme active site. The protein structure was further optimized via the protein preparation function in the MOE v2022. The structures of ligands were prepared in ChemDraw professional v16.0 and then it was imported to MOE for further optimization. After that, the ligands were docked using the online version of AutodockVina (v1.2.3) known as Webina53–55 (link) by creating a box with XYZ dimensions of 22 Å and around the catalytic hub of the modelled tyrosinase enzyme with the grid coordinates of the docking site centered at X = 32.154, Y = 140.176, Z = 215.585. The molecular interaction and conformational analysis of the ligand–protein complexes were performed in Biovia DS v2017 software.
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α-MSH is a peptide that is used in laboratory research. It is the principal endogenous melanocortin agonist and plays a role in pigmentation, energy homeostasis, and other physiological processes. α-MSH functions as a ligand for melanocortin receptors.
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L-DOPA is a laboratory product manufactured by Merck Group. It is a chemical compound used as a precursor in the synthesis of various pharmaceutical and research-related substances. The core function of L-DOPA is to serve as a starting material or intermediate in chemical reactions and processes. No further details or interpretations are provided.
More about "Tyrosinase-related protein-1"
Tyrosinase-related protein-1 (TYRP1), also known as brown locus protein or gp75, is a key enzyme involved in melanin biosynthesis and plays a crucial role in skin pigmentation and coloration.
As a member of the tyrosinase-related protein family, TYRP1 is essential for the proper synthesis and distribution of melanin, the pigment responsible for skin, hair, and eye color.
TYRP1 research is crucial for understanding the complex process of melanogenesis, the formation of melanin, as well as various pigmentation disorders and related dermatological conditions.
Understanding the expression, regulation, and function of TYRP1 can provide insights into conditions like albinism, vitiligo, and melasma.
Researchers investigating TYRP1 may utilize a variety of techniques and tools, such as TRIzol reagent for RNA extraction, FBS (fetal bovine serum) for cell culture, α-MSH (alpha-melanocyte-stimulating hormone) to stimulate melanogenesis, and PrimeScript RT reagent kit for reverse transcription.
DMSO (dimethyl sulfoxide) may be used as a solvent, while RIPA buffer is commonly employed for protein extraction and analysis.
Imaging techniques, such as the LSM 510 META confocal microscope, can be employed to visualize the localization and distribution of TYRP1 within cells.
Additionally, radioactive labeling with a 1900 CA scintillation analyzer or real-time PCR analysis using the 7500 Fast Real-Time PCR System may be utilized to study TYRP1 expression and activity.
The enzymatic activity of TYRP1 can also be assessed using L-DOPA (L-3,4-dihydroxyphenylalanine) as a substrate, which is a precursor in the melanin biosynthesis pathway.
By leveraging the insights and tools available for TYRP1 research, scientists can advance our understanding of pigmentation, develop new treatments for pigmentation disorders, and explore the broader implications of TYRP1 in various biological processes.
As a member of the tyrosinase-related protein family, TYRP1 is essential for the proper synthesis and distribution of melanin, the pigment responsible for skin, hair, and eye color.
TYRP1 research is crucial for understanding the complex process of melanogenesis, the formation of melanin, as well as various pigmentation disorders and related dermatological conditions.
Understanding the expression, regulation, and function of TYRP1 can provide insights into conditions like albinism, vitiligo, and melasma.
Researchers investigating TYRP1 may utilize a variety of techniques and tools, such as TRIzol reagent for RNA extraction, FBS (fetal bovine serum) for cell culture, α-MSH (alpha-melanocyte-stimulating hormone) to stimulate melanogenesis, and PrimeScript RT reagent kit for reverse transcription.
DMSO (dimethyl sulfoxide) may be used as a solvent, while RIPA buffer is commonly employed for protein extraction and analysis.
Imaging techniques, such as the LSM 510 META confocal microscope, can be employed to visualize the localization and distribution of TYRP1 within cells.
Additionally, radioactive labeling with a 1900 CA scintillation analyzer or real-time PCR analysis using the 7500 Fast Real-Time PCR System may be utilized to study TYRP1 expression and activity.
The enzymatic activity of TYRP1 can also be assessed using L-DOPA (L-3,4-dihydroxyphenylalanine) as a substrate, which is a precursor in the melanin biosynthesis pathway.
By leveraging the insights and tools available for TYRP1 research, scientists can advance our understanding of pigmentation, develop new treatments for pigmentation disorders, and explore the broader implications of TYRP1 in various biological processes.