Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease.

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.

In Situ Morphologic and Spectral Characterization of Retinal Pigment Epithelium Organelles in Mice Using Multicolor Confocal Fluorescence Imaging.

Purpose: To investigate the major organelles of the retinal pigment epithelium (RPE) in wild-type (WT, control) mice and their changes in pigmented Abca4 knockout (Abca4-/-) mice with in situ morphologic, spatial, and spectral characterization of live ex vivo flat-mounted RPE using multicolor confocal fluorescence microscopy (MCFM). Methods: In situ imaging of RPE flat-mounts of agouti Abca4-/- (129S4), agouti WT (129S1/SvlmJ) controls, and B6 albino mice (C57BL/6J-Tyrc-Brd) was performed with a Nikon A1 confocal microscope. High-resolution confocal image z-stacks of the RPE cell mosaic were acquired with four different excitation wavelengths (405 nm, 488 nm, 561 nm, and 640 nm). The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired and processed with Nikon NIS-AR Elements software. Results: The 3-dimensional multicolor confocal images provided a detailed visualization of the RPE cell mosaic, including its melanosomes and lipofuscin granules, and their varying characteristics in the different mice strains. The autofluorescence spectra, spatial distribution, and morphologic features of melanosomes and lipofuscin granules were measured. Increased numbers of lipofuscin and reduced numbers of melanosomes were observed in the RPE of Abca4-/- mice relative to controls. Conclusions: A detailed assessment of the RPE autofluorescent granules and their changes ex vivo was possible with MCFM. For all excitation wavelengths, autofluorescence from the RPE cells was predominantly contributed by lipofuscin granules, while melanosomes were found to be essentially nonfluorescent. The red shift of the emission peak confirmed the presence of multiple chromophores within lipofuscin granules. The elevated autofluorescence levels in Abca4-/- mice correlated well with the increased number of lipofuscin granules.

Defining an amyloid link Between Parkinson's disease and melanoma.

An epidemiological connection exists between Parkinson's disease (PD) and melanoma. α-Synuclein (α-syn), the hallmark pathological amyloid observed in PD, is also elevated in melanoma, where its expression is inversely correlated with melanin content. We present a hypothesis that there is an amyloid link between α-syn and Pmel17 (premelanosomal protein), a functional amyloid that promotes melanogenesis. Using SK-MEL 28 human melanoma cells, we show that endogenous α-syn is present in melanosomes, the organelle where melanin polymerization occurs. Using in vitro cross-seeding experiments, we show that α-syn fibrils stimulate the aggregation of a Pmel17 fragment constituting the repeat domain (RPT), an amyloidogenic domain essential for fibril formation in melanosomes. The cross-seeded fibrils exhibited α-syn-like ultrastructural features that could be faithfully propagated over multiple generations. This cross-seeding was unidirectional, as RPT fibrils did not influence α-syn aggregation. These results support our hypothesis that α-syn, a pathogenic amyloid, modulates Pmel17 aggregation in the melanosome, defining a molecular link between PD and melanoma.

Hierarchical biota-level and taxonomic controls on the chemistry of fossil melanosomes revealed using synchrotron X-ray fluorescence.

Fossil melanosomes, micron-sized granules rich in melanin in vivo, provide key information for investigations of the original coloration, taxonomy and internal anatomy of fossil vertebrates. Such studies rely, in part, on analysis of the inorganic chemistry of preserved melanosomes and an understanding of melanosome chemical taphonomy. The extent to which the preserved chemistry of fossil melanosomes is biased by biotic and abiotic factors is, however, unknown. Here we report the discovery of hierarchical controls on the inorganic chemistry of melanosomes from fossil vertebrates from nine biotas. The chemical data are dominated by a strong biota-level signal, indicating that the primary taphonomic control is the diagenetic history of the host sediment. This extrinsic control is superimposed by a biological, tissue-level control; tissue-specific chemical variation is most likely to survive in fossils where the inorganic chemistry of preserved melanosomes is distinct from that of the host sediment. Comparative analysis of our data for fossil and modern amphibians reveals that most fossil specimens show tissue-specific melanosome chemistries that differ from those of extant analogues, strongly suggesting alteration of original melanosome chemistry. Collectively, these findings form a predictive tool for the identification of fossil deposits with well-preserved melanosomes amenable to studies of fossil colour and anatomy.

Research Techniques Made Simple: Cell Biology Methods for the Analysis of Pigmentation.

Pigmentation of the skin and hair represents the result of melanin biosynthesis within melanosomes of epidermal melanocytes, followed by the transfer of mature melanin granules to adjacent keratinocytes within the basal layer of the epidermis. Natural variation in these processes produces the diversity of skin and hair color among human populations, and defects in these processes lead to diseases such as oculocutaneous albinism. While genetic regulators of pigmentation have been well studied in human and animal models, we are still learning much about the cell biological features that regulate melanogenesis, melanosome maturation, and melanosome motility in melanocytes, and have barely scratched the surface in our understanding of melanin transfer from melanocytes to keratinocytes. Herein, we describe cultured cell model systems and common assays that have been used by investigators to dissect these features and that will hopefully lead to additional advances in the future.

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.

Tissue-specific geometry and chemistry of modern and fossilized melanosomes reveal internal anatomy of extinct vertebrates.

Recent discoveries of nonintegumentary melanosomes in extant and fossil amphibians offer potential insights into the physiological functions of melanin not directly related to color production, but the phylogenetic distribution and evolutionary history of these internal melanosomes has not been characterized systematically. Here, we present a holistic method to discriminate among melanized tissues by analyzing the anatomical distribution, morphology, and chemistry of melanosomes in various tissues in a phylogenetically broad sample of extant and fossil vertebrates. Our results show that internal melanosomes in all extant vertebrates analyzed have tissue-specific geometries and elemental signatures. Similar distinct populations of preserved melanosomes in phylogenetically diverse vertebrate fossils often map onto specific anatomical features. This approach also reveals the presence of various melanosome-rich internal tissues in fossils, providing a mechanism for the interpretation of the internal anatomy of ancient vertebrates. Collectively, these data indicate that vertebrate melanins share fundamental physiological roles in homeostasis via the scavenging and sequestering of metals and suggest that intimate links between melanin and metal metabolism in vertebrates have deep evolutionary origins.

Effects of Aging on Hair Color, Melanosome Morphology, and Melanin Composition in Japanese Females.

In a previous study, we showed that the size of melanosomes isolated from Japanese female hairs enlarges with age, and this affects the hair color. In this study, we analyzed the age-dependent changes in hair melanin in order to further explore the factors related to hair color changing by aging. A significant positive correlation with age was found in the total melanin amount (TM) and the mol% of 5,6-dihydroxyindole (DHI) units, while no correlation was found in pheomelanin mol%. TM showed significant correlations with hair color parameters and the melanosome volume, suggesting that hair color darkening by aging is caused by the increase in TM due to the enlargement of the size of melanosome. From the measurement of absorbance spectra on synthetic eumelanins with different ratios of DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), we found that the increase in DHI mol% also contributes to the darkening of hair color by aging. In addition, the level of pyrrole-2,3-dicarboxylic acid (PDCA), a marker of DHI melanin, showed a significant negative correlation with the aspect ratio of melanosome, suggesting a contribution of DHI melanin to the change in melanosome morphology by aging.

Combined microscopy and spectroscopy techniques to characterize a fossilized feather with minimal damage to the specimen.

The study of fossil feathers has been revitalized in the last few decades and has contributed significantly to paleontological studies of dinosaurs and birds. Specific morphological and physicochemical characteristics of the microscale structures of feathers and the protein keratin are key targets when preserved during the fossilization process. Keratin is a fibrous protein that composes some hard tissues such as hair, nails and feathers. It is part of the so called intermediate filaments inside keratinocyte cells and is rich in sulfur containing amino acid cysteine. To date, different microscopy and analytical methods have been used for the analysis and detailed characterization and classification of feathers. However, in this work we showed that analytical optical and electron microscopies can be quick and precise methods with minimal effects on the sample during analysis. This association of different approaches on the same sample results in correlative data albeit in different length scales. Intracellular bodies called melanosomes originally present in melanocyte cells were identified with Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), and had well-defined orientation and a mean aspect ratio comparable to melanosomes extant in dark feathers. The detection of sulphur in melanosomes via Energy Dispersive Spectroscopy both in SEM and TEM shows that, along the fossilization process, sulphur from the degraded keratin matrix could have been trapped inside the melanosomes. Chemical groups that make up keratin and melanin in the fossil sample were detected via FT-IR Spectroscopy and Confocal Laser Scanning Microscopy (CLSM). The use of combined analytical microscopy techniques can contribute significantly to the study of fossils generating precise results with minimum damage to the original sample.

Melanosome diversity and convergence in the evolution of iridescent avian feathers-Implications for paleocolor reconstruction.

Some of the most varied colors in the natural world are created by iridescent nanostructures in bird feathers, formed by layers of melanin-containing melanosomes. The morphology of melanosomes in iridescent feathers is known to vary, but the extent of this diversity, and when it evolved, is unknown. We use scanning electron microscopy to quantify the diversity of melanosome morphology in iridescent feathers from 97 extant bird species, covering 11 orders. In addition, we assess melanosome morphology in two Eocene birds, which are the stem lineages of groups that respectively exhibit hollow and flat melanosomes today. We find that iridescent feathers contain the most varied melanosome morphologies of all types of bird coloration sampled to date. Using our extended dataset, we predict iridescence in an early Eocene trogon (cf. Primotrogon) but not in the early Eocene swift Scaniacypselus, and neither exhibit the derived melanosome morphologies seen in their modern relatives. Our findings confirm that iridescence is a labile trait that has evolved convergently in several lineages extending down to paravian theropods. The dataset provides a framework to detect iridescence with more confidence in fossil taxa based on melanosome morphology.

The chemical fingerprint of hair melanosomes by infrared nano-spectroscopy.

In situ characterization of the chemical and structural properties of black and white sheep hair was performed with a spatial resolution of 25 nm using infrared nano-spectroscopy. Comparing data sets from two types of hair allowed us to isolate the keratin FTIR fingerprint and so mark off chemical properties of the hair's melanosomes. From a polarization sensitive analysis of the nano-FTIR spectra, we showed that keratin intermediate filaments (IFs) present anisotropic molecular ordering. In stark contrast with white hair which does not contain melanosomes, in black hair, we spatially resolved single melanosomes and achieved unprecedented assignment of the vibrational modes of pheomelanin and eumelanin. The in situ experiment presented here avoids harsh chemical extractive methods used in previous studies. Our findings offer a basis for a better understanding of the keratin chemical and structural packing in different hair phenotypes as well as the involvement of melanosomes in hair color and biological functionality.

The Ultrastructure, Spatial Distribution, and Osmium Tetroxide Binding of Lipofuscin and Melanosomes in Aging Monkey Retinal Epithelium.

PURPOSE: To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology as a function of retinal eccentricity. METHODS: Electron microscopy was used to describe and quantify two major organelles in elderly monkey retinal epithelium, lipofuscin bodies and melanosomes, at different retinal loci extending from the macula to the peri-macula, equator, periphery and ora serrata. Osmium tetroxide was used to distinguish lipofuscin bodies from melanosomes. RESULTS: Lipofuscin bodies and melanosomes diminished in number with advanced age but there was an inverse relationship between these two organelles. Lipofuscin bodies were more numerous in the macula and melanosomes more numerous in the peripheral retina. Three types of lipofuscin bodies were identified: 1) smaller and tending to locate in the middle third of the epithelial cell, 2) larger, less common, and located more basally, and 3) extremely rare, melano-lipofuscin, containing a melanosome. When osmicated, all lipofuscin bodies contained electron dense materials. When osmium tetroxide was not used for fixation, the first two types of lipofuscin bodies lost their electron densities while the third type retained its electron density due to the melanosome it contained. CONCLUSION: As previously reported for human retina, lipofuscin is most abundant in the macular and peri-macular epithelium and least abundant in the periphery, whereas melanosomes show the opposite relationship. This distribution pattern could contribute to the macula's greater vulnerability to photo-toxicity. Three types of lipofuscin bodies are found in aging monkey retinal epithelium. All types contain electron dense material, but the most prominent two types lose their densities in the absence of osmium tetroxide during fixation. Most of the electron densities in lipofuscin bodies must contain a material that binds strongly to osmium tetroxide such as polyunsaturated fatty acids.

Melanosome-Targeting Near-Infrared Fluorescent Probe with Large Stokes Shift for in Situ Quantification of Tyrosinase Activity and Assessing Drug Effects on Differently Invasive Melanoma Cells.

Tyrosinase (TYR) plays a vital role in melanin biosynthesis and is widely regarded as a relatively specific marker for melanocytic lesions which involve vitiligo, malignant cutaneous melanoma, Parkinson's disease (PD), etc. However, the detection of TYR in living cells with fluorescent probes is usually interfered by diverse endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS). Herein, we synthesized a melanosome-targeting near-infrared (NIR) fluorescent probe (HB-NP) with a large Stokes shift (195 nm), achieving a highly sensitive and selective in situ detection for intracellular TYR, by incorporating a m-hydroxybenzyl moiety that recognizes TYR specifically and the morpholine unit which facilitates the probe accumulating in the melanosome into a salicyladazine skeleton. When treated with TYR, the probe itself with weak fluorescence is lit up via an inhibited photoinduced electron-transfer (PET) effect and HB-NP shows a strong fluorescence signal (nearly 48-fold enhancement) with a low detection limit of 0.5 U mL-1. HB-NP has been successfully applied in visualizing and in situ quantification of the intracellular TYR activity. Moreover, owing to the different expression levels of TYR, two human uveal melanoma cells with different invasive behaviors are distinguished by means of bioimaging and the effects of the inhibitor, kojic acid, and the up-regulating treatment, psoralen/ultraviolet A, on TYR activity of the two melanoma cells are evaluated. HB-NP is expected to be a useful tool to monitor diseases associated with the abnormal level of melanin and screen medicines for TYR disorder more effectively.

Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage.

Melanosomes are membrane-bound intracellular organelles that are uniquely generated by melanocytes (MCs) in the basal layer of human epidermis. Highly pigmented mature melanosomes are transferred from MCs to keratinocytes (KCs), and then positioned in the supra-nuclear region to ensure protection against ultraviolet radiation (UVR). However, the molecular mechanism underlying melanosome (or melanin pigment) transfer remains enigmatic. Emerging evidence shows that exo-/endo-cytosis of the melanosome core (termed melanocore) has been considered as the main transfer manner between MCs and KCs. As KCs in the skin migrate up from the basal layer and undergo terminal differentiation, the melanocores they have taken up from MCs are subjected to degradation. In this study, we isolated individual melanocores from human MCs in culture and then induced their destruction/disruption using a physical approach. The results demonstrate that the ultrastructural integrity of melanocores is essential for their antioxidant and photoprotective properties. In addition, we also show that cathepsin V (CTSV), a lysosomal acid protease, is involved in melanocore degradation in calcium-induced differentiated KCs and is also suppressed in KCs following exposure to UVA or UVB radiation. Thus, our study demonstrates that change in the proportion of melanocores in the intact/undegraded state by CTSV-related degradation in KCs affects photoprotection of the skin.

Magnificent magpie colours by feathers with layers of hollow melanosomes.

The blue secondary and purple-to-green tail feathers of magpies are structurally coloured owing to stacks of hollow, air-containing melanosomes embedded in the keratin matrix of the barbules. We investigated the spectral and spatial reflection characteristics of the feathers by applying (micro)spectrophotometry and imaging scatterometry. To interpret the spectral data, we performed optical modelling, applying the finite-difference time domain (FDTD) method as well as an effective media approach, treating the melanosome stacks as multi-layers with effective refractive indices dependent on the component media. The differently coloured magpie feathers are realised by adjusting the melanosome size, with the diameter of the melanosomes as well as their hollowness being the most sensitive parameters that influence the appearance of the feathers.

The nanomechanical role of melanin granules in the retinal pigment epithelium.

Nanomechanical properties of cells and tissues, in particular their elasticity, play an important role in different physiological and pathological processes. Recently, we have demonstrated that melanin granules dramatically modify nanomechanical properties of melanoma cells making them very stiff and, as a result, less aggressive. Although the mechanical effect of melanin granules was demonstrated in pathological cells, it was never studied in the case of normal cells. In this work, we analyzed the impact of melanin granules on nanomechanical properties of primary retinal pigment epithelium tissue fragments isolated from porcine eyes. The obtained results clearly show that melanin granules are responsible for the exceptional nanomechanical properties of the tissue. Our findings suggest that melanin granules in the retinal pigment epithelium may play an important role in sustaining the stiffness of this single cell layer, which functions as a natural mechanical barrier separating the retina from the choroid.

Novel organelles in primate retinal epithelium.

We are investigating age-related changes in organelles in monkey retinal epithelium using transmission and analytic electron microscopy. We previously described a circular organelle in retinal epithelium with a diameter of about 0.5µm. The organelle is unique in containing a single, round vacuole within an otherwise electron dense interior. We suggested that the organelle might be a melanosome with lysosomal properties. We now find that there are two similar organelles with such a single vacuole but which differ in their chemical composition, electron density, cell location and according to age. Epon embedded sections from the macular epithelium of seven monkeys, ranging from 1 to 35 years of age, were examined by transmission electron microscopy. A seven year old monkey was processed for analytic electron microscopy to determine the chemical composition of the organelles. The number and location of the organelles in the retinal epithelium were determined. The chemical composition of these two organelles was different. One of the organelles contained high mole fractions of oxygen and nitrogen and little phosphorous characteristic of melanin; the other had little oxygen and nitrogen and higher mole fractions of phosphorous uncharacteristic of melanin, but more common with lysosomal organelles. The latter had an electron dense rim around the vacuole, a less electron dense interior than the melanin containing organelle and also contained iron. The melanin containing organelle was more common in young monkeys and in the middle third of the cell. The organelle without melanin was more common in old monkeys and localized in the basal third of the cell. Two similarly vacuolated organelles, not identified before in retinal epithelium, differ in their chemical composition. One contains melanin; the other does not. The former is more common in young and the latter more common in old monkeys. This suggests reorganization and or degradation of melanin-containing organelles with age. These changes show how analytic electron microscopy can distinguish major ultra-structural differences in organelles when mere observation fails to do so easily.

TPC2 controls pigmentation by regulating melanosome pH and size.

Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

Bioinspired Artificial Melanosomes As Colorimetric Indicators of Oxygen Exposure.

Many industries require irreversibly responsive materials for use as sensors or detectors of environmental exposure. We describe the synthesis and fabrication of a nontoxic surface coating that reports oxygen exposure of the substrate material through irreversible formation of colored spots. The coating consists of a selectively permeable rubber film that contains the colorless organic precursors to darkly pigmented synthetic melanin. Melanin synthesis within the film is triggered by exposure to molecular oxygen. The selectively permeable rubber film regulates the rate of oxygen diffusion, enabling independent control of the sensitivity and response time of the artificial melanosome, while preventing leaching of melanin or its precursors.

A ß-solenoid model of the Pmel17 repeat domain: insights to the formation of functional amyloid fibrils.

Pmel17 is a multidomain protein involved in biosynthesis of melanin. This process is facilitated by the formation of Pmel17 amyloid fibrils that serve as a scaffold, important for pigment deposition in melanosomes. A specific luminal domain of human Pmel17, containing 10 tandem imperfect repeats, designated as repeat domain (RPT), forms amyloid fibrils in a pH-controlled mechanism in vitro and has been proposed to be essential for the formation of the fibrillar matrix. Currently, no three-dimensional structure has been resolved for the RPT domain of Pmel17. Here, we examine the structure of the RPT domain by performing sequence threading. The resulting model was subjected to energy minimization and validated through extensive molecular dynamics simulations. Structural analysis indicated that the RPT model exhibits several distinct properties of ß-solenoid structures, which have been proposed to be polymerizing components of amyloid fibrils. The derived model is stabilized by an extensive network of hydrogen bonds generated by stacking of highly conserved polar residues of the RPT domain. Furthermore, the key role of invariant glutamate residues is proposed, supporting a pH-dependent mechanism for RPT domain assembly. Conclusively, our work attempts to provide structural insights into the RPT domain structure and to elucidate its contribution to Pmel17 amyloid fibril formation.