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1.
PLoS Genet ; 19(4): e1010724, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37068079

RESUMO

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.


Assuntos
Catecol O-Metiltransferase , Galinhas , Camundongos , Animais , Galinhas/genética , Catecol O-Metiltransferase/genética , Camundongos Knockout , Melaninas/metabolismo , Pigmentação/genética , Mutação da Fase de Leitura
2.
Blood ; 125(10): 1623-32, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25477496

RESUMO

Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding diathesis, and other variable symptoms. The bleeding diathesis has been attributed to δ storage pool deficiency, reflecting the malformation of platelet dense granules. Here, we analyzed agonist-stimulated secretion from other storage granules in platelets from mouse HPS models that lack adaptor protein (AP)-3 or biogenesis of lysosome-related organelles complex (BLOC)-3 or BLOC-1. We show that α granule secretion elicited by low agonist doses is impaired in all 3 HPS models. High agonist doses or supplemental adenosine 5'-diphosphate (ADP) restored normal α granule secretion, suggesting that the impairment is secondary to absent dense granule content release. Intravital microscopy following laser-induced vascular injury showed that defective hemostatic thrombus formation in HPS mice largely reflected reduced total platelet accumulation and affirmed a reduced area of α granule secretion. Agonist-induced lysosome secretion ex vivo was also impaired in all 3 HPS models but was incompletely rescued by high agonist doses or excess ADP. Our results imply that (1) AP-3, BLOC-1, and BLOC-3 facilitate protein sorting to lysosomes to support ultimate secretion; (2) impaired secretion of α granules in HPS, and to some degree of lysosomes, is secondary to impaired dense granule secretion; and (3) diminished α granule and lysosome secretion might contribute to pathology in HPS.


Assuntos
Plaquetas/fisiologia , Síndrome de Hermanski-Pudlak/sangue , Complexo 3 de Proteínas Adaptadoras/deficiência , Complexo 3 de Proteínas Adaptadoras/genética , Complexo 3 de Proteínas Adaptadoras/fisiologia , Difosfato de Adenosina/farmacologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Degranulação Celular/fisiologia , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina , Síndrome de Hermanski-Pudlak/etiologia , Síndrome de Hermanski-Pudlak/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/deficiência , Lectinas/genética , Lectinas/fisiologia , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Selectina-P/sangue , Proteínas SNARE/sangue , Vesículas Secretórias/fisiologia , Trombina/farmacologia , Trombose/sangue , Trombose/etiologia , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiologia
3.
Blood ; 125(23): 3627-36, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25852052

RESUMO

Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application.


Assuntos
Plaquetas , Macrófagos , Megacariócitos , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Linhagem Celular , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/transplante , Camundongos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombopoese
4.
Blood ; 120(2): 404-14, 2012 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-22611153

RESUMO

Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.


Assuntos
Plaquetas/metabolismo , Síndrome de Hermanski-Pudlak/sangue , Síndrome de Hermanski-Pudlak/genética , Megacariócitos/metabolismo , Proteínas de Transporte de Monossacarídeos/sangue , Proteínas de Transporte de Monossacarídeos/genética , Animais , Plaquetas/patologia , Proteínas de Transporte/sangue , Diferenciação Celular , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA/sangue , Modelos Animais de Doenças , Endossomos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/sangue , Masculino , Megacariócitos/patologia , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Imunoeletrônica , Proteínas Mutantes/sangue , Proteínas Mutantes/genética , Proteínas Qa-SNARE/sangue , RNA Mensageiro/sangue , RNA Mensageiro/genética , Fatores de Transcrição/sangue
5.
Curr Biol ; 33(1): 86-97.e10, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36528024

RESUMO

Color variation is a frequent evolutionary substrate for camouflage in small mammals, but the underlying genetics and evolutionary forces that drive color variation in natural populations of large mammals are mostly unexplained. The American black bear, Ursus americanus (U. americanus), exhibits a range of colors including the cinnamon morph, which has a similar color to the brown bear, U. arctos, and is found at high frequency in the American southwest. Reflectance and chemical melanin measurements showed little distinction between U. arctos and cinnamon U. americanus individuals. We used a genome-wide association for hair color as a quantitative trait in 151 U. americanus individuals and identified a single major locus (p < 10-13). Additional genomic and functional studies identified a missense alteration (R153C) in Tyrosinase-related protein 1 (TYRP1) that likely affects binding of the zinc cofactor, impairs protein localization, and results in decreased pigment production. Population genetic analyses and demographic modeling indicated that the R153C variant arose 9.36 kya in a southwestern population where it likely provided a selective advantage, spreading both northwards and eastwards by gene flow. A different TYRP1 allele, R114C, contributes to the characteristic brown color of U. arctos but is not fixed across the range.


Assuntos
Ursidae , Animais , Fluxo Gênico , Variação Genética , Genoma , Estudo de Associação Genômica Ampla , Ursidae/genética
6.
Traffic ; 10(9): 1318-36, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19624486

RESUMO

Melanosomes are lysosome-related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky-Pudlak syndrome that lack BLOC-1, melanosomal proteins such as tyrosinase-related protein 1 (Tyrp1) accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here, we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverse early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT-I component, Tsg101, or inhibition of ESCRT function by dominant-negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT-dependent, ubiquitylated cargoes such as MART-1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101-depleted cells suggests that ESCRT-I functions downstream of BLOC-1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT-I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome-related organelle.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Microscopia de Fluorescência , Oxirredutases/biossíntese , Oxirredutases/genética , Fosfoproteínas/genética , Transporte Proteico , Fatores de Transcrição/genética , Transfecção
7.
Dev Cell ; 10(3): 343-54, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16516837

RESUMO

Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.


Assuntos
Endossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Organelas/metabolismo , Transporte Proteico , Vesículas Transportadoras/metabolismo , Antígenos de Neoplasias , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Antígeno MART-1 , Melanossomas/metabolismo , Glicoproteínas de Membrana/genética , Morfogênese , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Antígeno gp100 de Melanoma
8.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33886957

RESUMO

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex.


Assuntos
Complexo 3 de Proteínas Adaptadoras/genética , Subunidades delta do Complexo de Proteínas Adaptadoras/genética , Proteínas do Tecido Nervoso/genética , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/genética , Endossomos/genética , Humanos , Melanócitos/metabolismo , Melanossomas/genética , Transporte Proteico/genética
9.
J Invest Dermatol ; 140(2): 257-268.e8, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31980058

RESUMO

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.


Assuntos
Técnicas de Cultura de Células , Melaninas/análise , Melanossomas/química , Transtornos da Pigmentação/patologia , Pigmentação da Pele/fisiologia , Animais , Técnicas de Cocultura , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Queratinócitos/metabolismo , Melaninas/metabolismo , Melanossomas/metabolismo , Melanossomas/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Projetos de Pesquisa , Espectrofotometria/métodos
10.
J Cell Biol ; 161(3): 521-33, 2003 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-12732614

RESUMO

Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.


Assuntos
Células Eucarióticas/enzimologia , Glicoproteínas/metabolismo , Melanossomas/enzimologia , Microfibrilas/enzimologia , Proteínas/metabolismo , Subtilisinas/metabolismo , Compartimento Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Detergentes/farmacologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Células Eucarióticas/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Melaninas/metabolismo , Melanossomas/ultraestrutura , Glicoproteínas de Membrana , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Octoxinol/farmacologia , Peptídeo Hidrolases/metabolismo , Pró-Proteína Convertases , Estrutura Terciária de Proteína/fisiologia , Solubilidade/efeitos dos fármacos , Antígeno gp100 de Melanoma
11.
Mol Biol Cell ; 17(8): 3598-612, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16760433

RESUMO

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.


Assuntos
Endocitose , Retículo Endoplasmático/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação/genética , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células HeLa , Humanos , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Antígeno gp100 de Melanoma
12.
J Invest Dermatol ; 121(4): 821-30, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14632201

RESUMO

Pmel17 is a approximately 100 kDa pigment cell specific glycoprotein that plays a crucial part in the morphogenesis of melanosome precursors. Anti-Pmel17 immunoprecipitates from metabolically pulse labeled melanoma cells and melanocytes contain, in addition to full-length Pmel17, a glycoprotein that migrates with a lower relative molecular weight. Here we show that this glycoprotein is encoded by an mRNA that results from alternative splicing of the human Pmel17 gene from which a cryptic intron is excised. Immunoprecipitation recapture experiments showed that this glycoprotein contained both the N- and C-termini of full-length Pmel17. Sequence analysis of cDNA corresponding to the alternatively spliced form reveals the loss of three of 10 imperfect direct repeats from the central region of the lumenal domain. The product of the splice variant is processed with similar kinetics to full-length Pmel17, and localizes similarly to late endosomes when expressed ectopically in nonpigment cells. We speculate that truncation of the repeat region within Pmel17 alters either fibrillogenic activity or the interaction of Pmel17 with melanin intermediates. The expression of an alternatively spliced product may furthermore affect the cohort of peptides generated for recognition of melanoma cells by tumor-directed T lymphocytes.


Assuntos
Melanócitos/fisiologia , Melanoma , Proteínas/genética , Splicing de RNA , Neoplasias Cutâneas , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Epitopos , Humanos , Melaninas/metabolismo , Melanócitos/citologia , Melanossomas/fisiologia , Glicoproteínas de Membrana , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/imunologia , RNA Mensageiro/genética , Linfócitos T/imunologia , Sequências de Repetição em Tandem , Antígeno gp100 de Melanoma
13.
Pigment Cell Melanoma Res ; 26(4): 470-86, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23452376

RESUMO

Proteolytic fragments of the pigment cell-specific glycoprotein, PMEL, form the amyloid fibrillar matrix underlying melanins in melanosomes. The fibrils form within multivesicular endosomes to which PMEL is selectively sorted and that serve as melanosome precursors. GPNMB is a tissue-restricted glycoprotein with substantial sequence homology to PMEL, but no known function, and was proposed to localize to non-fibrillar domains of distinct melanosome subcompartments in melanocytes. Here we confirm that GPNMB localizes to compartments distinct from the PMEL-containing multivesicular premelanosomes or late endosomes in melanocytes and HeLa cells, respectively, and is largely absent from fibrils. Using domain swapping, the unique PMEL localization is ascribed to its polycystic kidney disease (PKD) domain, whereas the homologous PKD domain of GPNMB lacks apparent sorting function. The difference likely reflects extensive modification of the GPNMB PKD domain by N-glycosylation, nullifying its sorting function. These results reveal the molecular basis for the distinct trafficking and morphogenetic properties of PMEL and GPNMB and support a deterministic function of the PMEL PKD domain in both protein sorting and amyloidogenesis.


Assuntos
Amiloide/química , Endossomos/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/química , Antígeno gp100 de Melanoma/química , Linhagem Celular Tumoral , DNA Complementar/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Células HeLa , Humanos , Melaninas/química , Melanócitos/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/metabolismo
14.
Mol Biol Cell ; 20(5): 1464-77, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19116314

RESUMO

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes.


Assuntos
Melanossomas/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Motivos de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Glicosilação , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Pigmentos Biológicos/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia
15.
J Biol Chem ; 284(16): 10877-89, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19240024

RESUMO

Melanoregulin (MREG), the product of the Mreg(dsu) gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg(-/-) mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg(-/-) mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Epitélio Pigmentado Ocular/citologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteínas de Transporte/genética , Catepsina D/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipofuscina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/fisiologia , Fosfatidiletanolaminas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Compostos de Piridínio/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Retinaldeído/análogos & derivados , Retinaldeído/metabolismo , Retinoides/metabolismo
16.
J Biol Chem ; 283(4): 2307-22, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-17991747

RESUMO

Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.


Assuntos
Amiloide/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Amiloide/genética , Anticorpos/química , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/genética , Endossomos/ultraestrutura , Epitopos/genética , Epitopos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Melanossomas/genética , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Antígeno gp100 de Melanoma
17.
J Cell Sci ; 116(Pt 21): 4441-54, 2003 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-13130094

RESUMO

tGolgin-1 (golgin-245, trans golgi p230) and golgin-97 are members of a family of peripheral membrane proteins of unknown function that localize to the trans Golgi network (TGN) through a conserved C-terminal GRIP domain. We have probed for GRIP protein function by assessing the consequences of overexpressing isolated GRIP domains. By semi-quantitative immunofluorescence microscopy we found that high level expression of epitope-tagged, GRIP domain-containing fragments of tGolgin-1 or golgin-97 specifically altered the characteristic pericentriolar distribution of TGN integral membrane and coat components. Concomitantly, vesicular transport from the TGN to the plasma membrane and furin-dependent cleavage of substrate proteins in the TGN were inhibited. Mutagenesis of a conserved tyrosine in the tGolgin-1 GRIP domain abolished these effects. GRIP domain overexpression had little effect on the distribution of most Golgi stack resident proteins and no effect on markers of other organelles. Electron microscopy analyses of GRIP domain-overexpressing cells revealed distended perinuclear vacuoles and a proliferation of multivesicular late endosomes to which the TGN resident protein TGN46 was largely mislocalized. These studies, the first to address the function of GRIP domain-containing proteins in higher eukaryotes, suggest that some or all of these proteins and/or their ligands function in maintaining the integrity of the TGN by regulating resident protein localization.


Assuntos
Autoantígenos/metabolismo , Endossomos/metabolismo , Rede trans-Golgi/metabolismo , Clonagem Molecular , Glicoproteínas/metabolismo , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Ligantes , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína
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