RESUMEN
Understanding how natural selection acts on the genome and contributes to the process of speciation is a primary aim of the study of evolution. Here we used natural variation in two subspecies of the Guadeloupean anole (Anolis marmoratus ssp.), from the island of Guadeloupe in the Lesser Antilles, to explore the genomic basis of adaptation and speciation in Anolis lizards. These subspecies inhabit distinct ecological environments and display marked differences in adult male color and pattern. We sequenced the complete genomes of 20 anoles, 10 from each subspecies, at 1.4× coverage. We used genome-wide scans of population differentiation, allele frequency spectrum, and linkage disequilibrium to characterize the genomic architecture within and between the subspecies. While most of the genome was undifferentiated, we observed five large divergent regions. Within these regions we identified blocks, 5 kb pairs in length, enriched for fixed single nucleotide polymorphisms. These blocks encompass 97 genes, two of which are candidate pigmentation genes. One is melanophilin (mlph), which helps transport melanosomes within melanocytes. The other is a cluster of differentiation 36 (cd36), which regulates carotenoid pigment sequestration. We used high-pressure liquid chromatography to confirm that carotenoid pigments are significantly more abundant in the conspicuous orange-pigmented skin of male A. m. marmoratus suggesting that cd36 may be regulating pigment deposition in this tissue. We identified for the first time a carotenoid gene that is a potential target of divergent sexual selection and may be contributing to the early stages of speciation in Anolis lizards.
RESUMEN
16-kauren-2-beta-18, 19-triol (16-kauren) is a natural diterpenoid substance derived from Asteraceae psiadia punctulata, a small tropical shrub in Africa and Asia, and it can reduce Mlph expression without affecting the expression of Rab27a and MyoVa in melanocytes. Melanophilin (Mlph) is an important linker protein in the melanosome transport process. However, the signal transduction pathway for the regulation of Mlph expression has not been fully established. We examined the mechanism of 16-kauren on Mlph expression. Murine melan-a melanocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, and luciferase assay were performed. The inhibition of Mlph expression by 16-kauren-2ß-18,19-triol (16-kauren) occurs through the JNK signal and is reversed following glucocorticoid receptor (GR) activation by dexamethasone (Dex). Especially, 16-kauren activates JNK and c-jun signalling, part of the MAPK pathway, with subsequent Mlph repression. When the JNK signal is weakened by siRNA, the inhibition of Mlph expression by 16-kauren was not seen. JNK activation by 16-kauren induces GR phosphorylation, which leads to Mlph repression. These results demonstrate that 16-kauren regulates Mlph expression through the phosphorylation of GR via the JNK signalling pathway.
Asunto(s)
Melanocitos , Receptores de Glucocorticoides , Ratones , Animales , Receptores de Glucocorticoides/metabolismo , Fosforilación , Melanocitos/metabolismo , Melanosomas/metabolismo , Transporte Biológico , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Exon skipping is an efficient technique to inhibit specific gene expression induced by a short-sequence peptide nucleic acid (PNA). To date, there has been no study on the effects of PNA on skin pigmentation. In melanocytes, the tripartite complex is responsible for the transport of mature melanosomes from the nucleus to the dendrites. The tripartite complex is composed of Rab27a, Mlph (Melanophilin), and Myosin Va. Defects in the protein Mlph, a melanosome transport-related protein, are known to cause hypopigmentation. Our study shows that Olipass peptide nucleic acid (OPNA), a cell membrane-permeable PNA, targets exon skipping in the Mlph SHD domain, which is involved in Rab27a binding. Our findings demonstrate that OPNA induced exon skipping in melan-a cells, resulting in shortened Mlph mRNA, reduced Mlph protein levels, and melanosome aggregation, as observed by microscopy. Therefore, OPNA inhibits the expression of Mlph by inducing exon skipping within the gene. These results suggest that OPNA, which targets Mlph, may be a potential new whitening agent to inhibit melanosome movement.
RESUMEN
In mammals, the main contribution to the variability of pigmentation is made by two groups of genes directly related to the metabolic pathways of pigment synthesis and controlling the transport of melanosomes in melanocytes to keratinocytes. In order to identify the genetic basis of pigmentation variants, the nucleotide sequences of the melanophilin gene were compared in two groups of ferrets-silver-colored and wild-type animals-using sequencing of 16 exons. In carriers of silver color, a single nucleotide deletion was detected in the 9th exon, leading to a shift in the reading frame and the formation of a stop codon downstream. The protein encoded by the mutant allele is almost completely devoid of the C terminal domain of the protein responsible for the contact of melanosomes with actin during their moving to the periphery of melanocytes, but it retains the leading domain involved in the formation of melanosomes. The combination of the preservation of the N domain and the defect of the C domain of the mutant protein for the first time makes it possible to explain the incomplete dominance of the wild-type protein in heterozygotes.
Asunto(s)
Hurones , Plata , Animales , Hurones/genética , Plata/metabolismo , Melanocitos/metabolismo , Melanosomas/genética , Melanosomas/metabolismo , ExonesRESUMEN
BACKGROUND: Melanosomes are intracellularly transported from the perinuclear region to the cell periphery and then to neighboring keratinocytes. We recently reported that the flavonoid rhamnazin suppresses melanosomal transport within pigment cells, yet the action mechanism remained unclear. OBJECTIVE: Our aim was to elucidate how rhamnazin influences the intracellular transport of melanosomes. METHODS: A melanosome distribution assay and immunostaining were performed using B16F10 mouse melanoma cells and normal human epidermal melanocytes, respectively. Expression levels of melanosome transport-related proteins, including melanophilin (MLPH), RAB27A, and myosin VA (MYO5A), were analyzed by immunoblotting. Ubiquitinated MLPH was detected using a commercial ubiquitin detection kit. To investigate the interaction between rhamnazin and MLPH, we prepared rhamnazin conjugated with magnetic FG beads. RESULTS: Immunoblotting analysis revealed that rhamnazin specifically reduces the expression of MLPH but not RAB27A or MYO5A proteins. The ubiquitin detection assay, which made use of a proteasome inhibitor, showed that MLPH accumulated as a polyubiquitinated protein after treatment with rhamnazin. We speculated that the affinity of rhamnazin for the components of the melanosome transport-related tripartite complex may alter the stability of the formation of the tripartite assembly. By using affinity-based techniques with B16F10 whole cell lysates or recombinant MLPH and RAB27A proteins, we revealed the interaction of rhamnazin with the components of the tripartite complex. CONCLUSION: We found that rhamnazin inhibits intracellular transport of melanosomes through proteasomal degradation of MLPH. Our results suggest that topical application of rhamnazin may provide a new approach for treating skin pigmentation disorders.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Flavonoles/farmacología , Melanosomas/efectos de los fármacos , Pigmentación de la Piel/efectos de los fármacos , Animales , Línea Celular Tumoral , Células Cultivadas , Evaluación Preclínica de Medicamentos , Flavonoles/uso terapéutico , Humanos , Hiperpigmentación/tratamiento farmacológico , Melaninas/biosíntesis , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Data mining of a public transcriptomic rectal cancer dataset (GSE35452) from the Gene Expression Omnibus, National Center for Biotechnology Information identified the melanophilin (MLPH) gene as the most significant intracellular protein transport-related gene (GO:0006886) associated with a poor response to preoperative chemoradiation. An MLPH immunostain was performed on biopsy specimens from 172 rectal cancer patients receiving preoperative chemoradiation; samples were divided into high- and low-expression groups by H-scores. Subsequently, the correlations between MLPH expression and clinicopathologic features, tumor regression grade, disease-specific survival (DSS), local recurrence-free survival (LRFS), and metastasis-free survival (MeFS) were analyzed. MLPH expression was significantly associated with CEA level (p = 0.001), pre-treatment tumor status (p = 0.022), post-treatment tumor status (p < 0.001), post-treatment nodal status (p < 0.001), vascular invasion (p = 0.028), and tumor regression grade (p < 0.001). After uni- and multi-variable analysis of five-year survival, MLPH expression was still associated with lower DSS (hazard ratio (HR), 10.110; 95% confidence interval (CI), 2.178-46.920; p = 0.003) and MeFS (HR, 5.621; 95% CI, 1.762-17.931; p = 0.004). In conclusion, identifying MLPH expression could help to predict the response to chemoradiation and survival, and aid in personal therapeutic modification.
RESUMEN
Altered melanosome transport in melanocytes, resulting from variants in the melanophilin (MLPH) gene, are associated with inherited forms of coat color dilution in many species. In dogs, the MLPH gene corresponds to the D locus and two variants, c.-22G > A (d1) and c.705G > C (d2), leading to the dilution of coat color, as described. Here, we describe the independent investigations of dogs whose coat color dilution could not be explained by known variants, and who report a third MLPH variant, (c.667_668insC) (d3), which leads to a frameshift and premature stop codon (p.His223Profs*41). The d3 allele is found at low frequency in multiple dog breeds, as well as in wolves, wolf-dog hybrids, and indigenous dogs. Canids in which the d3 allele contributed to the grey (dilute) phenotype were d1/d3 compound heterozygotes or d3 homozygotes, and all non-dilute related dogs had one or two D alleles, consistent with a recessive inheritance. Similar to other loci responsible for coat colors in dogs, this, alongside likely additional allelic heterogeneity at the D locus, or other loci, must be considered when performing and interpreting genetic testing.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Color , Color del Cabello/genética , Pigmentación/genética , Alelos , Animales , Codón sin Sentido/genética , Perros , Exones/genética , Mutación del Sistema de Lectura/genética , Homocigoto , Humanos , FenotipoRESUMEN
Prohibitin (PHB, also known as PHB1 or BAP32), is a highly conserved 31kDa protein that expressed in many cellular compartments, such as mitochondria, nucleus, cytosol, and plasma membrane, and plays roles in regulating the transcription of genes, apoptosis, and mitochondrial biogenesis. There is a report that Prohibitin expression is required for the stimulation of pigmentation by melanogenin. However, no studies have been published on the function of PHB in melanocytes, especially in melanosome transport. Methods: Immunofluorescence was performed to confirm the localization of PHB. siRNA transfections, Co-immunoprecipitation, western blotting and proximity ligation assay were performed to find binding state between proteins and demonstrate functions of PHB on melanosome transport. Results: PHB is located in the melanosome and perinuclear aggregation of melanosome is induced when expression of PHB is reduced with no influence on melanin contents. PHB binds directly to Rab27a and Mlph but not Myosin-Va. Rab27a and Mlph bind to specific domains of PHB. Reduced expression of PHB led to the impaired binding affinity between Rab27a and Mlph. Conclusion: PHB regulates melanosome transport by linking to Rab27a and Mlph in melanocytes. Targeting and regulating PHB not only manages pigmentation in melanocytes, but also controls hyperpigmentation in melanoma.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Melaninas/metabolismo , Melanosomas/metabolismo , Proteínas Represoras/fisiología , Proteínas rab27 de Unión a GTP/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Pigmentación , Prohibitinas , Unión ProteicaRESUMEN
BACKGROUND: Rab27a, Mlph, and MyoVa form a tripartite complex and relate to melanosome distribution. Melanophilin (Mlph) acts as a linker protein between Rab27a and MyoVa. The biological activity and function of 16-kauren on the expression of Mlph has not yet been studied. OBJECTIVE: We examined the effect of 16-kauren on melanosome transport and skin pigmentation. METHODS: Murine Melan-a melanocytes and SP-1 keratinocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, luciferase assay and immunohistochemical staining in 3D pigmented human skin model were performed. RESULTS: We found that 16-kauren inhibits melanosome transport in Melan-a melanocytes without affecting melanin synthesis. Treatment with 16-kauren reduced melanophilin (Mlph), a key protein in melanosome transport, in Melan-a melanocytes, at both the protein and mRNA levels while it did not affect the expression of Rab27a and MyoVa, the other two key proteins for melanosome transport. Notably, the expression of melanogenic proteins, including tyrosinase, trp1, trp2, and MITF, was not affected by 16-kauren. However, 16-kauren attenuated melanosome distribution in co-culture of Melan-a melanocytes and SP-1 keratinocytes as well as in Melan-a monolayer culture. In further confirmation of the depigmenting effects of 16-kauren on Melanoderm™, a 3D pigmented human skin model, treatment with 16-kauren for 12 days increased the brightness of the tissue as determined by lightness value and reduced the distribution of melanosomes as shown in histological examination. CONCLUSION: These results demonstrated that 16-kauren is a selective modulator of a melangenic target, Mlph expression, and can be employed as a new depigmenting strategy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diterpenos de Tipo Kaurano/farmacología , Melanocitos/efectos de los fármacos , Melanosomas/efectos de los fármacos , Pigmentación de la Piel/efectos de los fármacos , Animales , Asteraceae/química , Línea Celular Tumoral , Técnicas de Cocultivo , Regulación hacia Abajo/efectos de los fármacos , Queratinocitos , Antígeno MART-1/genética , Antígeno MART-1/metabolismo , Melaninas/biosíntesis , Melanocitos/citología , Melanocitos/metabolismo , Melanosomas/metabolismo , RatonesRESUMEN
Melanosomes undergo a complex maturation process and migrate into keratinocytes. Melanophilin (Mlph), a protein complex involving myosin Va (MyoVa) and Rab27a, enables the movement of melanosomes in melanocytes. In this study, we found six miRNAs targeting Mlph in mouse using two programs (http://targetscan.org and DianaTools). When melan-a melanocytes were treated with six synthesized microRNAs, miR-342-5p, miR-1839-5p, and miR-3082-5p inhibited melanosome transport and induced melanosome aggregation around the nucleus. The other microRNAs, miR-5110, miR-3090-3p, and miR-186-5p, did not inhibit melanosome transport. Further, miR-342-5p, miR-1839-5p, and miR-3082-5p decreased Mlph expression. The effect of miR-342-5p was the strongest among the six synthesized miRNAs. It inhibited melanosome transport in melan-a melanocytes and reduced Mlph expression in mRNA and protein levels in a dose-dependent manner; however, it did not affect Rab27a and MyoVa expressions, which are associated with melanosome transport. To examine miR-342-5p specificity, we performed luciferase assays in a mouse melanocyte-transfected reporter vector including Mlph at the 3'-UTR (untranslated region). When treated with miR-342-5p, luciferase activity that had been reduced by approximately 50% was restored after inhibitor treatment. Therefore, we identified a novel miRNA affecting Mlph and melanosome transport, and these results can be used for understanding Mlph expression and skin pigmentation regulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Melanosomas/metabolismo , MicroARNs/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , MicroARNs/genéticaRESUMEN
Vertebrate myosin-5a is an ATP-utilizing processive motor associated with the actin network and responsible for the transport and localization of several vesicle cargoes. To transport cargo efficiently and prevent futile ATP hydrolysis, myosin-5a motor function must be tightly regulated. The globular tail domain (GTD) of myosin-5a not only functions as the inhibitory domain but also serves as the binding site for a number of cargo adaptor proteins, including melanophilin (Mlph) and Rab-interacting lysosomal protein-like 2 (RILPL2). In this study, using various biochemical approaches, including ATPase, single-molecule motility, GST pulldown assays, and analytical ultracentrifugation, we demonstrate that the binding of both Mlph and RILPL2 to the GTD of myosin-5a is required for the activation of myosin-5a motor function under physiological ionic conditions. We also found that this activation is regulated by the small GTPase Rab36, a binding partner of RILPL2. In summary, our results indicate that RILPL2 is required for Mlph-mediated activation of Myo5a motor activity under physiological conditions and that Rab36 promotes this activation. We propose that Rab36 stimulates RILPL2 to interact with the myosin-5a GTD; this interaction then induces exposure of the Mlph-binding site in the GTD, enabling Mlph to interact with the GTD and activate myosin-5a motor activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Motoras Moleculares/fisiología , Miosina Tipo V/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Ratones , Proteínas Motoras Moleculares/metabolismo , Miosina Tipo V/metabolismo , Concentración Osmolar , Unión ProteicaRESUMEN
Spitzoid neoplasms typically affect young individuals and include Spitz nevus, atypical Spitz tumor, and Spitzoid melanoma. Spitz tumors can exhibit gene fusions involving the receptor tyrosine kinases NTRK1, NTRK3, ALK, ROS1, RET, or MET, or the serine-threonine kinase BRAF. Because most studies have been based on adult cases, we studied ALK fusions in Spitz nevi occurring in pediatric patients. Twenty-seven cases were screened for ALK expression by immunohistochemistry, and 6 positive cases were identified. These cases were studied further using the TruSight RNA Fusion Panel, and in 4 cases, exon 20 of the ALK gene was found to be fused to exon 14 of the MLPH (melanophilin) gene, a gene fusion that has only been reported in a Spitz nevus in an adult. The remaining 2 cases showed no fusion of ALK with any gene. The cases with the MLPH-ALK fusion showed a similar histology to that described for Spitz nevi with ALK fusions, with spindle-shaped and epithelioid melanocytes in fusiform nests with a plexiform growth pattern and infiltrative border. We created a breakapart fluorescence in situ hybridization assay for MLPH, and all 4 cases with the MLPH-ALK fusion were positive, whereas the other 23 cases in the study were negative. Thus, ALK and MLPH were fused only to each other in our series. Melanophilin is part of the melanosome trafficking apparatus together with MYO5a, TPM3, and RAB27a, all constitutively expressed in melanocytes. Kinase fusions involving MYO5A and TPM3 have been reported in Spitz tumors, and our series adds MLPH to this group.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Quinasa de Linfoma Anaplásico/genética , Nevo de Células Epitelioides y Fusiformes/genética , Neoplasias Cutáneas/genética , Adolescente , Niño , Preescolar , Femenino , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Nevo de Células Epitelioides y Fusiformes/patología , Fusión de Oncogenes , Neoplasias Cutáneas/patologíaRESUMEN
Melanophilin (Mlph) forms an interaction with Rab27a and the actin-based motor protein MyosinVa (MyoVa) on mature melanosome membranes and the tripartite complex regulates melanosome transport in melanocytes. In this study, we found that Rab27a siRNA decreased Mlph and Rab27a protein levels, but Mlph mRNA levels were not changed. Other Rab27a siRNA sequences also showed the same results. When Rab27a siRNA was treated with melan-a melanocytes, Rab27a protein was decreased within 6 hours and Mlph protein was decreased within 24 hours. To determine whether the absence of Rab27a promotes Mlph degradation, we inhibited protein degradation by treatment with proteasome (MG132) and lysosomal enzyme (E64D and Pepstatin A) inhibitors in melan-a melanocytes. MG132 inhibited the degradation of Mlph, but E64D and Pepstatin A had no effect on Mlph. The absence of Rab27a enhanced ubiquitination of Mlph and induced proteasomal degradation. From these results, we concluded that Mlph interaction with Rab27a is important for Mlph stability and melanosome transport.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transporte Biológico/genética , Proteínas rab27 de Unión a GTP/genética , Proteínas rab27 de Unión a GTP/metabolismo , Animales , Línea Celular , Antígeno MART-1/metabolismo , Melanocitos/metabolismo , Melanosomas/fisiología , Ratones , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacologíaRESUMEN
Coat color dilution corresponds to a specific pigmentation phenotype that leads to a dilution of wild type pigments. It affects both eumelanin and pheomelanin containing melanosomes. The mode of inheritance of the dilution phenotype is autosomal recessive. Candidate gene approaches focused on the melanophilin (MLPH) gene highlighted two variants associated with the dilution phenotype in rabbits: The c.111-5C>A variant that is located in an acceptor splice site or the c.585delG variant, a frameshift mutation. On the transcript level, the skipping of two exons has been reported as the molecular mechanism responsible for the coat color dilution. To clarify, which of the two variants represents the causal variant, (i) we analyzed their allelic segregation by genotyping Castor and Chinchilla populations, and (ii) we evaluated their functional effects on the stability of MLPH transcripts in skin samples of animals with diluted or wild type coat color. Firstly, we showed that the c.585delG variant showed perfect association with the dilution phenotype in contrast to the intronic c.111-5C>A variant. Secondly, we identified three different MLPH isoforms including the wild type isoform, the exon-skipping isoform and a retained intron isoform. Thirdly, we observed a drastic and significant decrease of MLPH transcript levels in rabbits with a coat color dilution (p-values ranging from 10-03 to 10-06). Together, our results bring new insights into the coat color dilution trait.
RESUMEN
Frequent kinase fusions have been reported in spitzoid neoplasms, approximately 10% of which involve ALK rearrangements. Herein, we report a case of atypical Spitz tumor with a novel MLPH-ALK fusion, which has not been previously reported to contribute to cancer development. The tumor was detected in the right arm of a 40-year-old woman. The novel ALK fusion was identified by a 5'-rapid amplification of cDNA ends-based system optimized for formalin-fixed, paraffin-embedded tissue. Initially, ALK expression was detected by immunohistochemistry using 5A4 antibodies for both sensitive and conventional polymer detection methods. However, the anti-ALK1 antibody, which is commonly used for the diagnosis of ALK-positive anaplastic large cell lymphoma, failed to confirm ALK expression. These results indicated that ALK immunohistochemistry results in ALK-rearranged atypical Spitz tumor may differ based on the type of primary antibody clone, which can be a potential diagnostic pitfall.
Asunto(s)
Quinasa de Linfoma Anaplásico/metabolismo , Nevo de Células Epitelioides y Fusiformes/patología , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias Cutáneas/patología , Adulto , Quinasa de Linfoma Anaplásico/genética , Femenino , Humanos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Nevo de Células Epitelioides y Fusiformes/diagnóstico , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias Cutáneas/genéticaRESUMEN
Pigment organelles, or melanosomes, are transported by kinesin, dynein, and myosin motors. As such, melanosome transport is an excellent model system to study the functional relationship between the microtubule- and actin-based transport systems. In mammalian melanocytes, it is well known that the Rab27a/melanophilin/myosin Va complex mediates actin-based transport in vivo. However, pathways that regulate the overall directionality of melanosomes on the actin/microtubule networks have not yet been delineated. Here, we investigated the role of PKA-dependent phosphorylation on the activity of the actin-based Rab27a/melanophilin/myosin Va transport complex in vitro. We found that melanophilin, specifically its C-terminal actin-binding domain (ABD), is a target of PKA. Notably, in vitro phosphorylation of the ABD closely recapitulated the previously described in vivo phosphorylation pattern. Unexpectedly, we found that phosphorylation of the ABD affected neither the interaction of the complex with actin nor its movement along actin tracks. Surprisingly, the phosphorylation state of melanophilin was instead important for reversible association with microtubules in vitro. Dephosphorylated melanophilin preferred binding to microtubules even in the presence of actin, whereas phosphorylated melanophilin associated with actin. Indeed, when actin and microtubules were present simultaneously, melanophilin's phosphorylation state enforced track selection of the Rab27a/melanophilin/myosin Va transport complex. Collectively, our results unmasked the regulatory dominance of the melanophilin adaptor protein over its associated motor and offer an unexpected mechanism by which filaments of the cytoskeletal network compete for the moving organelles to accomplish directional transport on the cytoskeleton in vivo.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Microtúbulos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Colorantes Fluorescentes , Melanocitos/metabolismo , Melanosomas/metabolismo , Ratones , Modelos Biológicos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Fosforilación , Dominios Proteicos , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas rab27 de Unión a GTP/genética , Proteínas rab27 de Unión a GTP/metabolismoRESUMEN
Soon after RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease opened a new era of targeted genome editing, the CRISPR/Cas9 platform began to be extensively used to modify genes in various types of cells and organisms. However, successful CRISPR/Cas9-mediated insertion/deletion (indel) mutation remains to be demonstrated in avian cell lines. The objective of this study was to design a poultry-specific CRISPR/Cas9 system to efficiently introduce targeted deletion mutation in chromosomes of the quail muscle clone 7 (QM7) cell line using a customized quail CRISPR vector. In this study, two avian-specific promoters, quail 7SK (q7SK) promoter and CBh promoter, the hybrid form of cytomegalovirus and chicken ß-actin promoters, were cloned into a CRISPR vector for the expression of guide RNA and Cas9 protein, respectively. Then, guide RNA, which was designed to target 20-base pair (bp) nucleotides in the quail melanophilin (MLPH) locus, was ligated to the modified CRISPR vector and transfected to QM7 cells. Our results showed multiple indel mutations in the quail MLPH locus in nearly half of the alleles being tested, suggesting the high efficiency of the system for targeted gene modification. The new CRISPR vector developed from this study has the potential application to generate knockout avian cell lines and knockout poultry.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/veterinaria , Marcación de Gen/métodos , Codorniz/genética , Animales , Línea Celular , Edición Génica/métodos , Mutación INDEL , Edición de ARN/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
Genome-wide association studies have identified genomic loci, whose single-nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin-immunoprecipitation-coupled sequencing and microarray expression profiling in TMPRSS2-ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor-binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR-binding motif, which is enriched in the neighborhood of canonical androgen-responsive elements. TâG exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor-suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Elementos de Respuesta , Adulto , Anciano , Alelos , Secuencia de Bases , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Neoplasias de la Próstata/patología , Unión Proteica , Carga TumoralRESUMEN
BACKGROUND: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare and often fatal disorder characterized by defective cellular cytotoxicity and hyperinflammation, and the only cure known to date is hematopoietic stem cell transplantation. Mutations in RAB27A, LYST, and AP3B1 give rise to FHL associated with oculocutaneous albinism, and patients with FHL are usually only screened for mutations in these genes when albinism is observed. A number of patients with FHL and normal pigmentation remain without a genetic diagnosis. OBJECTIVE: We asked whether patients with FHL with immunodeficiency but with normal pigmentation might sometimes have mutations that affected cellular cytotoxicity without affecting pigmentation. METHODS: We carried out mutation analysis of RAB27A, LYST, and AP3B1 in patients with FHL with pigment dilution, as well as a cohort with no clinical evidence of pigment dilution but no mutations in the other known FHL-related genes (PRF1, STXBP2, and UNC13D). RESULTS: We identify patients with Griscelli syndrome type 2 with biallelic mutations in RAB27A in the absence of albinism. All 6 patients carried mutations at amino acids R141, Y159, or S163 of Rab27a that disrupt the interaction of Rab27a with Munc13-4, without impairing the interaction between melanophilin and Rab27a. CONCLUSION: These studies highlight the need for RAB27A sequencing in patients with FHL with normal pigmentation and identify a critical binding site for Munc13-4 on Rab27a, revealing the molecular basis of this interaction.
Asunto(s)
Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Pigmentación de la Piel/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Albinismo/genética , Estudios de Casos y Controles , Degranulación de la Célula , Línea Celular , Niño , Preescolar , Estudios de Cohortes , Citotoxicidad Inmunológica , Análisis Mutacional de ADN , Femenino , Expresión Génica , Marcadores Genéticos , Humanos , Lactante , Linfohistiocitosis Hemofagocítica/diagnóstico , Masculino , Proteínas de la Membrana/química , Modelos Moleculares , Perforina/genética , Fenotipo , Unión Proteica , Conformación Proteica , Proteínas de Unión al GTP rab/química , Proteínas rab27 de Unión a GTPRESUMEN
We investigated the inhibitory effects of hesperidin on melanogenesis. To find melanosome transport inhibitor from natural products, we collected the structural information of natural products from Korea Food and Drug Administration (KFDA) and performed pharmacophore-based in silico screening for Rab27A and melanophilin (MLPH). Hesperidin did not inhibit melanin production in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH), and also did not affect the catalytic activity of tyrosinase. But, hesperidin inhibited melanosome transport in melanocyte and showed skin lightening effect in pigmented reconstructed epidermis model. Therefore, we suggest that hesperidin is a useful inhibitor of melanosome transport and it might be applied to whitening agent.