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The Journal of Biological Chemistry Oct 2023Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously...
Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here, we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous Parkinson's disease-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38-binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.
Topics: Animals; Mice; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Melanocytes; Mutation; Parkinson Disease; Phosphorylation; rab GTP-Binding Proteins; HEK293 Cells; Humans; Gene Expression; Protein Domains; Protein Binding; Intracellular Membranes
PubMed: 37625589
DOI: 10.1016/j.jbc.2023.105192 -
Neoplasia (New York, N.Y.) Sep 2023Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is...
Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.
Topics: Humans; Melanoma; Skin Neoplasms; Carcinoma, Squamous Cell; Lysosomes; rab GTP-Binding Proteins; Melanoma, Cutaneous Malignant
PubMed: 37562257
DOI: 10.1016/j.neo.2023.100924 -
Science (New York, N.Y.) Aug 2023Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering...
Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering properties of melanin to conduct a genome-wide screen for regulators of melanogenesis. We identified 169 functionally diverse genes that converge on melanosome biogenesis, endosomal transport, and gene regulation, of which 135 represented previously unknown associations with pigmentation. In agreement with their melanin-promoting function, the majority of screen hits were up-regulated in melanocytes from darkly pigmented individuals. We further unraveled functions of KLF6 as a transcription factor that regulates melanosome maturation and pigmentation in vivo, and of the endosomal trafficking protein COMMD3 in modulating melanosomal pH. Our study reveals a plethora of melanin-promoting genes, with broad implications for human variation, cell biology, and medicine.
Topics: Humans; Melanins; Melanocytes; Melanosomes; Skin Pigmentation; Genome-Wide Association Study; Adaptor Proteins, Signal Transducing; Kruppel-Like Factor 6; Endosomes; Animals; Mice; Cell Line, Tumor
PubMed: 37561850
DOI: 10.1126/science.ade6289 -
Theranostics 2023Senescent melanocytes accumulate in photoaged skin and are closely related to skin aging. A better understanding of the molecular characteristics of senescent...
Senescent melanocytes accumulate in photoaged skin and are closely related to skin aging. A better understanding of the molecular characteristics of senescent melanocytes may be the key to controlling skin aging. We have developed an model of senescence in melanocytes using UV irradiation and investigated the functional characteristics and molecular mechanisms underlying senescence in UV-irradiated melanocytes. We have highlighted that senescent melanocytes are characterized by melanosome transport dysfunction resulting in melanin accumulation. The defective melanosome transport was confirmed with the ultrastructural characterization of both UV-induced senescent melanocytes and melanocytes of hypopigmented aging skin. A single-cell transcriptomic analysis revealed that the glycolytic metabolism pathway appeared to be significantly upregulated in most senescent phenotypes. Furthermore, the inhibition of glycolysis by pharmacological compounds mitigates the pro-aging effects of melanocytes senescence, suggesting that alterations in cellular glucose metabolism act as a driving force for senescence in melanocytes. These results demonstrate that senescent melanocytes are characterized by glycolytic metabolism changes and a defective melanosome transport process, which may be related to impaired mitochondrial function, highlighting the importance of metabolic reprogramming in regulating melanocyte senescence.
Topics: Melanosomes; Melanocytes; Skin; Melanins; Glycolysis; Cellular Senescence
PubMed: 37554281
DOI: 10.7150/thno.84912 -
International Journal of Molecular... Jul 2023Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding... (Review)
Review
Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding keratinocytes. While the molecular mechanisms of melanin synthesis and transport in melanocytes are now well characterized, much less is known about melanin transfer and processing within keratinocytes. Over the past few decades, distinct models have been proposed to explain how melanin transfer occurs at the cellular and molecular levels. However, this remains a debated topic, as up to four different models have been proposed, with evidence presented supporting each. Here, we review the current knowledge on the regulation of melanin exocytosis, internalization, processing, and polarization. Regarding the different transfer models, we discuss how these might co-exist to regulate skin pigmentation under different conditions, i.e., constitutive and facultative skin pigmentation or physiological and pathological conditions. Moreover, we discuss recent evidence that sheds light on the regulation of melanin exocytosis by melanocytes and internalization by keratinocytes, as well as how melanin is stored within these cells in a compartment that we propose be named the melanokerasome. Finally, we review the state of the art on the molecular mechanisms that lead to melanokerasome positioning above the nuclei of keratinocytes, forming supranuclear caps that shield the nuclear DNA from UV radiation. Thus, we provide a comprehensive overview of the current knowledge on the molecular mechanisms regulating skin pigmentation, from melanin exocytosis by melanocytes and internalization by keratinocytes to processing and polarization within keratinocytes. A better knowledge of these molecular mechanisms will clarify long-lasting questions in the field that are crucial for the understanding of skin pigmentation and can shed light on fundamental aspects of organelle biology. Ultimately, this knowledge can lead to novel therapeutic strategies to treat hypo- or hyper-pigmentation disorders, which have a high socio-economic burden on patients and healthcare systems worldwide, as well as cosmetic applications.
Topics: Humans; Melanins; Melanocytes; Keratinocytes; Epidermis; Skin Pigmentation; Melanosomes
PubMed: 37511054
DOI: 10.3390/ijms241411289 -
Ecology and Evolution Jul 2023Understanding 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...
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 ( ssp.), from the island of Guadeloupe in the Lesser Antilles, to explore the genomic basis of adaptation and speciation in 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 (), which helps transport melanosomes within melanocytes. The other is a cluster of differentiation 36 (), 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 . . suggesting that 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 lizards.
PubMed: 37435022
DOI: 10.1002/ece3.10266 -
Bioscience Reports Jul 2023Recent innovations in computational structural biology have opened an opportunity to revise our current understanding of the structure and function of clinically...
Recent innovations in computational structural biology have opened an opportunity to revise our current understanding of the structure and function of clinically important proteins. This study centres on human Oca2 which is located on mature melanosomal membranes. Mutations of Oca2 can result in a form of oculocutanous albinism, which is the most prevalent and visually identifiable form of albinism. Sequence analysis predicts Oca2 to be a member of the SLC13 transporter family, but it has not been classified into any existing SLC families. The modelling of Oca2 with AlphaFold2 and other advanced methods show that, like SLC13 members, it consists of a scaffold and transport domain and displays a pseudo inverted repeat topology that includes re-entrant loops. This finding contradicts the prevailing consensus view of its topology. In addition to the scaffold and transport domains, the presence of a cryptic GOLD domain is revealed that is likely responsible for its trafficking from the endoplasmic reticulum to the Golgi prior to localisation at the melanosomes. The GOLD domain harbours some known glycosylation sites. Analysis of the putative ligand binding site of the model shows the presence of highly conserved key asparagine residues that suggest Oca2 may be a Na+/dicarboxylate symporter. Known critical pathogenic mutations map to structural features present in the repeat regions that form the transport domain. Exploiting the AlphaFold2 multimeric modelling protocol in combination with conventional homology modelling allowed the building of plausible homodimers in both inward- and outward-facing conformations, supporting an elevator-type transport mechanism.
Topics: Humans; Membrane Transport Proteins; Albinism, Oculocutaneous; Mutation
PubMed: 37431738
DOI: 10.1042/BSR20230060 -
The Journal of Biological Chemistry Aug 2023Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid...
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.
Topics: Humans; Melanosomes; Monophenol Monooxygenase; Niemann-Pick C1 Protein; Cholesterol; Niemann-Pick Diseases; Niemann-Pick Disease, Type C
PubMed: 37423302
DOI: 10.1016/j.jbc.2023.105024