RESUMEN
Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.
Asunto(s)
Glaucoma de Ángulo Abierto , Enfermedades Neurodegenerativas , Animales , Humanos , Adulto Joven , Amiloide/metabolismo , Ojo/metabolismo , Glaucoma de Ángulo Abierto/metabolismo , Antígeno gp100 del Melanoma/genética , Melanosomas/genética , Melanosomas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Pez CebraRESUMEN
Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.
Asunto(s)
Enfermedad de Niemann-Pick Tipo C , Enfermedades de Niemann-Pick , Humanos , Melanosomas/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteína Niemann-Pick C1/metabolismo , Colesterol/metabolismo , Enfermedades de Niemann-Pick/genética , Enfermedades de Niemann-Pick/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismoRESUMEN
The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.
Asunto(s)
Melanocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutación , Enfermedades Neurodegenerativas/patología , Antígeno gp100 del Melanoma/metabolismo , Secuencia de Aminoácidos , Proteínas Amiloidogénicas/metabolismo , Animales , Biología Computacional/métodos , Humanos , Glicoproteínas de Membrana/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Filogenia , Pigmentación , Dominios Proteicos , Homología de Secuencia , Antígeno gp100 del Melanoma/genéticaRESUMEN
The biosynthesis pathway of melanin is a series of oxidative reactions that are catalyzed by melanin-related proteins, including tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). Reagents or materials with antioxidative or free radical-scavenging activities may be candidates for anti-melanogenesis. 3,4-Dihydroxybenzalacetone (DBL) is a polyphenol isolated from fungi, such as Phellinus obliguus (Persoon) Pilat and P. linteus. In this study, we investigated the effects and mechanisms of DBL on antioxidation and melanogenesis in murine melanoma cells (B16F10) and human epidermal melanocytes (HEMs). The results indicated that DBL scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals, and exhibited potent reducing power, indicating that it displays strong antioxidative activity. DBL also inhibited the expression of TYR, TRP-1, TRP-2, and microphthalmia-related transcription factor (MITF) in both the cells. In addition, DBL inhibited hyperpigmentation in B16F10 and HEMs by regulating the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), v-akt murine thymoma viral oncogene homolog (AKT)/glycogen synthase kinase 3 beta (GSK3ß), and mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinase (ERK) signaling pathways. DBL not only shortened dendritic melanocytes but also inhibited premelanosome protein 17 (PMEL17) expression, slowing down the maturation of melanosome transportation. These results indicated that DBL promotes anti-melanogenesis by inhibiting the transportation of melanosomes. Therefore, DBL is a potent antioxidant and depigmenting agent that may be used in whitening cosmetics.
Asunto(s)
Ácidos Cafeicos/farmacología , Regulación hacia Abajo/efectos de los fármacos , Epidermis/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanocitos/metabolismo , Melanosomas/metabolismo , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanosomas/genéticaRESUMEN
The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar ß-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a "nucleator" of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.
Asunto(s)
Empalme Alternativo , Amiloide/química , Agregado de Proteínas , Antígeno gp100 del Melanoma/química , Amiloide/genética , Amiloide/metabolismo , Humanos , Isoformas de Proteínas , Estructura Secundaria de Proteína , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Lipids often play an important role in the initial steps of fibrillation. The melanosomal protein Pmel17 forms amyloid in vivo and contains a highly amyloidogenic Repeat domain (RPT), important for melanin biosynthesis. RPT fibrillation is influenced by two lysolipids, the anionic lysophosphatidylglycerol (LPG) and zwitterionic lysophosphatidylcholine (LPC), both present in vivo at elevated concentrations in melanosomes, organelles in which Pmel17 aggregate. Here we investigate the interaction of RPT with both LPG and LPC using small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), electron microscopy, fluorescence and circular dichroism (CD) spectroscopy. Under non-shaking conditions, both lipids promote fibrillation but this is driven by different interactions with RPT. Each RPT binds >40 LPG molecules but only weak interactions are seen with LPC. Above LPG's criticial micelle concentration (cmc), LPG and RPT form connected micelles where RPT binds to the surface as beads on a string with core-shell structures. Binding to LPG only induces α-helical structure well above the cmc, while LPC has no measurable effect on the protein structure. While low (but still super-cmc) concentrations of LPG strongly promote aggregation, at higher LPG concentrations (10â¯mM), only ~ one RPT binds per micelle, inhibiting amyloid formation. ITC and SAXS reveal some interactions between the zwitterionic lipid LPC and RPT below the cmc but little above the cmc. Nevertheless, LPC only promotes aggregation above the cmc and this process is not inhibited by high LPC concentrations, suggesting that monomers and micelles cooperate to influence amyloid formation.
Asunto(s)
Amiloide/química , Lisofosfatidilcolinas/química , Lisofosfolípidos/química , Antígeno gp100 del Melanoma/química , Agregación Patológica de Proteínas , Dominios ProteicosRESUMEN
Prions have served as pathfinders that reveal many aspects of proteostasis in neurons. The recent realization that several prominent neurodegenerative diseases spread via a prion-like mechanism illuminates new possibilities for diagnostics and therapeutics. Thus, key proteins in Alzheimer Disease and Amyotrophic lateral sclerosis (ALS), including amyloid-ß precursor protein, Tau and superoxide dismutase 1 (SOD1), spread to adjacent cells in their misfolded aggregated forms and exhibit template-directed misfolding to induce further misfolding, disruptions to proteostasis and toxicity. Here we invert this comparison to ask what these prion-like diseases can teach us about the broad prion disease class, especially regarding the loss of these key proteins' function(s) as they misfold and aggregate. We also consider whether functional amyloids might reveal a role for subverted protein function in neurodegenerative disease. Our synthesis identifies SOD1 as an exemplar of protein functions being lost during prion-like protein misfolding, because SOD1 is inherently unstable and loses function in its misfolded disease-associated form. This has under-appreciated parallels amongst the canonical prion diseases, wherein the normally folded prion protein, PrPC, is reduced in abundance in fatal familial insomnia patients and during the preclinical phase in animal models, apparently via proteostatic mechanisms. Thus while template-directed misfolding and infectious properties represent gain-of-function that fascinates proteostasis researchers and defines (is required for) the prion(-like) diseases, loss and subversion of the functions attributed to hallmark proteins in neurodegenerative disease needs to be integrated into design towards effective therapeutics. We propose experiments to uniquely test these ideas.
Asunto(s)
Enfermedades por Prión/patología , Proteínas Priónicas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Humanos , Enfermedades por Prión/metabolismo , Proteínas Priónicas/química , Pliegue de Proteína , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismo , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
Considering the hidden mode of inheritance of some coat-color-associated alleles, we investigated the presence/absence of coat-color-associated alleles in 1093 domestic horses of 55 breeds and 20 specimens of Przewalski's horse. For coat-color genotyping, allele specific PCR, pyrosequencing and Li-Cor analyses were conducted on 12 coat-color-associated alleles of five genes. Our data provide deep insight into the distribution of coat-color-associated alleles within breeds. We found that the alleles for the basic colorations (bay, black, and chestnut) are widely distributed and occur in nearly all breeds. Alleles leading to dilutions or patterns are rare in domestic breeds and were not found in Przewalski's horse. Higher frequencies of these alleles are only found in breeds that are selected for their expressed phenotypes (e.g., Kinsky horse, Lewitzer, Tinker). Nevertheless, our study produced strong evidence that molecular testing of the coat color is necessary for well-defined phenotyping to avoid unexpected colorations of offspring that can result in legal action.
Asunto(s)
Genética de Población , Cabello , Caballos/genética , Pigmentación/genética , Alelos , Animales , Cruzamiento , Técnicas de Genotipaje , Caballos/clasificación , FenotipoRESUMEN
The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils.
Asunto(s)
Amiloide/química , Modelos Moleculares , Cuerpos Multivesiculares/ultraestructura , Procesamiento Proteico-Postraduccional , Antígeno gp100 del Melanoma/química , Sustitución de Aminoácidos , Amiloide/metabolismo , Amiloide/ultraestructura , Línea Celular Tumoral , Cisteína/química , Cisteína/metabolismo , Cistina/química , Cistina/metabolismo , Dimerización , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Kringles , Microscopía Electrónica de Transmisión , Peso Molecular , Cuerpos Multivesiculares/química , Cuerpos Multivesiculares/metabolismo , Mutagénesis Sitio-Dirigida , Mutación Puntual , Pliegue de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/metabolismoRESUMEN
BACKGROUND: ADAMs (a disintegrin and metalloprotease) are a family of proteases involved in ectodomain shedding that play a role in various biological processes such as cell adhesion and migration. ADAM10 and ADAM17 are suggested to be involved in pigmentary disorders. OBJECTIVE: We examined the effect of ADAM protease inhibitors on the modulation of melanogenesis in normal human epidermal melanocytes (NHEM). METHODS: NHEMs and B16F10 treated with ADAM protease inhibitors were analyzed. AlamarBlue cell proliferation assay, melanin content assay, tyrosinase activity assay, Western blotting analysis, electron microscopic analysis, and RNA interference were employed. RESULTS: In NHEMs, melanin content was reduced by treatment with ADAM protease inhibitors. The inhibitors did not change the protein expression of tyrosinase, TRP-1, and MITF. In B16F10 cells, treatment of the cells with ADAM protease inhibitor diminished the α-MSH-induced increase in melanin content. Electron microscopy showed that the number of fibrillar and mature melanosomes was significantly reduced and that the vacuolar compartments were filled with dense unstructured aggregates after treatment with ADAM protease inhibitors. We therefore focused on the processing of PMEL17, a melanosomal glycoprotein that forms a fibrillar matrix on which melanin gets deposited. Proteolytic processing of PMEL17 is required to form functional fibrils during melanogenesis. Recently, γ-secretase and ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2) were found to cleave PMEL17. We found that ADAM protease inhibitors exerted effects on the processing of C-terminal and N-terminal fragments of PMEL17. Using BACE2 siRNA and γ-secretase inhibitor, we showed that ADAM protease inhibitor affected PMEL17 processing in a γ-secretase and BACE2-independent mechanism. CONCLUSION: Several proteases, including ADAM proteases, can contribute to the formation of fibrils and their assembly into sheets in melanosomes.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Melaninas/biosíntesis , Melanocitos/metabolismo , Melanoma Experimental/metabolismo , Inhibidores de Proteasas/farmacología , Antígeno gp100 del Melanoma/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Línea Celular Tumoral , Dipéptidos/farmacología , Humanos , Interferón Tipo I/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/ultraestructura , Melanoma Experimental/ultraestructura , Melanosomas/efectos de los fármacos , Melanosomas/ultraestructura , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microscopía Electrónica , Monofenol Monooxigenasa/metabolismo , Proteínas Gestacionales/metabolismo , ARN Interferente Pequeño/farmacología , alfa-MSH/metabolismoRESUMEN
Fibrils derived from Pmel17 are functional amyloids upon which melanin is deposited. Fibrils of the repeat domain (RPT) of Pmel17 form under strict melanosomal pH (4.5-5.5) and completely dissolve at pH≥6. To determine which Glu residue is responsible for this reversibility, aggregation of single, double, and quadruple Ala and Gln mutants were examined by intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy. Charge neutralization of E404, E422, E425, or E430, which are located in the putative amyloid-forming region, modulated aggregation kinetics. Remarkably, the removal of a single negative charge at E422, one of 16 carboxylic acids, shifted the pH dependence by a full pH unit. Mutation at E404, E425, or E430 had little to no effect. We suggest that protonation at E422 is essential for initiating amyloid formation and that the other Glu residues play an allosteric role in fibril stability.
Asunto(s)
Amiloide/metabolismo , Amiloide/química , Concentración de Iones de Hidrógeno , Cinética , Tamaño de la Partícula , Conformación Proteica , Propiedades de SuperficieRESUMEN
The characterisation of the pleiotropic effects of coat colour-associated mutations in mammals illustrates that sensory organs and nerves are particularly affected by disorders because of the shared origin of melanocytes and neurocytes in the neural crest; e.g. the eye-colour is a valuable indicator of disorders in pigment production and eye dysfunctions. Disorders related to coat colour-associated alleles also occur in the skin (melanoma), reproductive tract and immune system. Additionally, the coat colour phenotype of an individual influences its general behaviour and fitness. Mutations in the same genes often produce similar coat colours and pleiotropic effects in different species (e.g., KIT [reproductive disorders, lethality], EDNRB [megacolon] and LYST [CHS]). Whereas similar disorders and similar-looking coat colour phenotypes sometimes have a different genetic background (e.g., deafness [EDN3/EDNRB, MITF, PAX and SNAI2] and visual diseases [OCA2, RAB38, SLC24A5, SLC45A2, TRPM1 and TYR]). The human predilection for fancy phenotypes that ignore disorders and genetic defects is a major driving force for the increase of pleiotropic effects in domestic species and laboratory subjects since domestication has commenced approximately 18,000 years ago.