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Mitsunori Fukuda - One of the best experts on this subject based on the ideXlab platform.
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rab gtpases key players in melanosome biogenesis transport and transfer
Pigment Cell & Melanoma Research, 2020Co-Authors: Mitsunori FukudaAbstract:Melanosomes are specialized intracellular organelles that produce and store melanin pigments in melanocytes, which are present in several mammalian tissues and organs, including the skin, hair, and eyes. Melanosomes form and mature stepwise (stages I-IV) in melanocytes and then are transported toward the plasma membrane along the cytoskeleton. They are subsequently transferred to neighboring keratinocytes by a largely unknown mechanism, and incorporated melanosomes are transported to the perinuclear region of the keratinocytes where they form melanin caps. Melanocytes also extend several dendrites that facilitate the efficient transfer of the melanosomes to the keratinocytes. Since the melanosome biogenesis, transport, and transfer steps require multiple membrane trafficking processes, Rab GTPases that are conserved key regulators of membrane traffic in all eukaryotes are crucial for skin and hair pigmentation. Dysfunctions of two Rab isoforms, Rab27A and Rab38, are known to cause a hypopigmentation phenotype in human type 2 Griscelli Syndrome patients and in chocolate mice (related to Hermansky-Pudlak Syndrome), respectively. In this review article, I review the literature on the functions of each Rab isoform and its upstream and downstream regulators in mammalian melanocytes and keratinocytes.
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The GTPase-deficient Rab27A(Q78L) mutant inhibits melanosome transport in melanocytes through trapping of Rab27A effector protein Slac2-a/melanophilin in their cytosol: development of a novel melanosome-targetinG tag.
The Journal of biological chemistry, 2014Co-Authors: Morié Ishida, Saki P. Arai, Norihiko Ohbayashi, Mitsunori FukudaAbstract:The small GTPase Rab27A is a crucial regulator of actin-based melanosome transport in melanocytes, and functionally defective Rab27A causes human Griscelli Syndrome type 2, which is characterized by silvery hair. A GTPase-deficient, constitutively active Rab27A(Q78L) mutant has been shown to act as an inhibitor of melanosome transport and to induce perinuclear aggregation of melanosomes, but the molecular mechanism by which Rab27A(Q78L) inhibits melanosome transport remained to be determined. In this study, we attempted to identify the primary cause of the perinuclear melanosome aggregation induced by Rab27A(Q78L). The results showed that Rab27A(Q78L) is unable to localize on mature melanosomes and that its inhibitory activity on melanosome transport is completely dependent on its binding to the Rab27A effector Slac2-a/melanophilin. When we forcibly expressed Rab27A(Q78L) on mature melanosomes by using a novel melanosome-targeting tag that we developed in this study and named the MST tag, the MST-Rab27A(Q78L) fusion protein behaved in the same manner as wild-type Rab27A. It localized on mature melanosomes without inducing melanosome aggregation and restored normal peripheral melanosome distribution in Rab27A-deficient cells. These findings indicate that the GTPase activity of Rab27A is required for its melanosome localization but is not required for melanosome transport.
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Functional characterization of two RAB27A missense mutations found in Griscelli Syndrome type 2.
Pigment cell & melanoma research, 2010Co-Authors: Norihiko Ohbayashi, Mitsunori Fukuda, Setareh Mamishi, Koutaro Ishibashi, Yuto Maruta, Babak Pourakbari, Banafshe Tamizifar, Masoud Mohammadpour, Nima ParvanehAbstract:Human Griscelli Syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.
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Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases.
Structure (London England : 1993), 2008Co-Authors: Mutsuko Kukimoto-niino, Mitsunori Fukuda, Ayako Sakamoto, Eiko Kanno, Kyoko Hanawa-suetsugu, Takaho Terada, Mikako Shirouzu, Shigeyuki YokoyamaAbstract:Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli Syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.
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Functional analysis of slac2-a/melanophilin as a linker protein between Rab27A and myosin Va in melanosome transport.
Methods in enzymology, 2005Co-Authors: Taruho S. Kuroda, Takashi Itoh, Mitsunori FukudaAbstract:Slac2-a/melanophilin regulates melanosome transport in mammalian skin melanocytes by linking melanosome-bound Rab27A and an actin-based motor protein, myosin Va. Slac2-a consists of an N-terminal Slp homology domain (SHD), which has been identified as a specific GTP-Rab27-binding domain, a myosin Va-binding domain (MBD) in the middle region, and an actin-binding domain (ABD) at the C-terminus. Mutations in the slac2-a/mlph gene cause the abnormal pigmentation (i.e., perinuclear melanosome aggregation in melanocytes) in human Griscelli Syndrome type III and in leaden mice because of the inability to form the tripartite protein complex consisting of Rab27A, Slac2-a, and myosin Va. In this chapter we describe the methods, including in vivo melanosome distribution assay combined with dominant-negative approaches and RNA interference technology, that have been used to analyze the function of Slac2-a in melanosome transport in melanocytes.
Kathleen Freson - One of the best experts on this subject based on the ideXlab platform.
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Rab proteins and Rab-associated proteins: major actors in the mechanism of protein-trafficking disorders
European Journal of Pediatrics, 2008Co-Authors: Lucien Corbeel, Kathleen FresonAbstract:Ras-associated binding (Rab) proteins and Rab-associated proteins are key regulators of vesicle transport, which is essential for the delivery of proteins to specific intracellular locations. More than 60 human Rab proteins have been identified, and their function has been shown to depend on their interaction with different Rab-associated proteins regulating Rab activation, post-translational modification and intracellular localization. The number of known inherited disorders of vesicle trafficking due to Rab cycle defects has increased substantially during the past decade. This review describes the important role played by Rab proteins in a number of rare monogenic diseases as well as common multifactorial human ones. Although the clinical phenotype in these monogenic inherited diseases is highly variable and dependent on the type of tissue in which the defective Rab or its associated protein is expressed, frequent features are hypopigmentation (Griscelli Syndrome), eye defects (Choroideremia, Warburg Micro Syndrome and Martsolf Syndrome), disturbed immune function (Griscelli Syndrome and Charcot–Marie–Tooth disease) and neurological dysfunction (X-linked non-specific mental retardation, Charcot–Marie–Tooth disease, Warburg Micro Syndrome and Martsolf Syndrome). There is also evidence that alterations in Rab function play an important role in the progression of multifactorial human diseases, such as infectious diseases and type 2 diabetes. Rab proteins must not only be bound to GTP, but they need also to be ‘prenylated’—i.e. bound to the cell membranes by isoprenes, which are intermediaries in the synthesis of cholesterol (e.g. geranyl geranyl or farnesyl compounds). This means that isoprenylation can be influenced by drugs such as statins, which inhibit isoprenylation, or biphosphonates, which inhibit that farnesyl pyrophosphate synthase necessary for Rab GTPase activity. Conclusion : Although protein-trafficking disorders are clinically heterogeneous and represented in almost every subspeciality of pediatrics, the identification of common pathogenic mechanisms may provide a better diagnosis and management of patients with still unknown Rab cycle defects and stimulate the development of therapeutic agents.
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REVIEW Rab proteins and Rab-associated proteins: major actors in the mechanism of protein-trafficking disorders
2008Co-Authors: Lucien Corbeel, Kathleen FresonAbstract:Abstract Ras-associated binding (Rab) proteins and Rabassociated proteins are key regulators of vesicle transport, which is essential for the delivery of proteins to specific intracellular locations. More than 60 human Rab proteins have been identified, and their function has been shown to depend on their interaction with different Rab-associated proteins regulating Rab activation, post-translational modification and intracellular localization. The number of known inherited disorders of vesicle trafficking due to Rab cycle defects has increased substantially during the past decade. This review describes the important role played by Rab proteins in a number of rare monogenic diseases as well as common multifactorial human ones. Although the clinical phenotype in these monogenic inherited diseases is highly variable and dependent on the type of tissue in which the defective Rab or its associated protein is expressed, frequent features are hypopigmentation (Griscelli Syndrome), eye defects (Choroideremia, Warburg Micro Syndrome and Martsolf Syndrome), disturbed immune function (Griscelli Syndrome and Charcot–Marie–Tooth disease) and neurological dysfunction (X-linked non-specific mental retardation, Charcot–Marie–Tooth disease, Warburg Micro Syndrome and Martsolf Syndrome). There is also evidence that alterations in Rab function play an important role in the progression of multifactorial human diseases, such as infectious diseases and type 2 diabetes. Rab proteins must no
Jo Lambert - One of the best experts on this subject based on the ideXlab platform.
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Griscelli Syndrome: a model system to study vesicular trafficking.
Pigment cell & melanoma research, 2009Co-Authors: Mireille Van Gele, Peter Dynoodt, Jo LambertAbstract:Griscelli Syndrome (GS) is a rare autosomal recessive disorder caused by mutations in either the myosin VA (GS1), RAB27A (GS2) or melanophilin (GS3) genes. The three GS subtypes are commonly characterized by pigment dilution of the skin and hair, due to defects involving melanosome transport in melanocytes. Here, we review how detailed studies concerning GS have contributed to a better understanding of the molecular mechanisms involved in vesicle transport and membrane trafficking processes. Additionally, we demonstrate that the identification and biological analysis of novel disease-causing mutations highlighted the functional importance of the RAB27A-MLPH-MYO5A tripartite complex in intracellular melanosome transport. As the small GTPase Rab27a is able to interact with multiple effectors, including Slp2-a and Myrip, we report on their presumed role in melanosome transport. Furthermore, we summarize data suggesting that RAB27B and RAB27A are functionally redundant and hereby provide further insight into the pathogenesis of GS2. Finally, we discuss how the gathered knowledge about the RAB27A-MLPH-MYO5A tripartite complex can be translated into a possible therapeutic application to reduce (hyper)pigmentation of the skin.
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Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli Syndrome
The Journal of biological chemistry, 2003Co-Authors: Philippe Bahadoran, Mitsunori Fukuda, Roser Buscà, Karine Bille, Christine Chiaverini, Wendy Westbroek, Jo Lambert, Gaëlle Valony, Jean-marie Naeyaert, Jean-paul OrtonneAbstract:Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli Syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.
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Arg–Cys Substitution at Codon 1246 of the Human Myosin Va Gene is not Associated with Griscelli Syndrome
The Society for Investigative Dermatology Inc., 2000Co-Authors: Jo Lambert, Naeyaert, Jean Marie, De Paepe Anne, Van Coster Rudy, Ferster Alina, Song Micheline, Messiaen LudwineAbstract:Myosin Va is an actin-associated motor protein involved in organelle transport such as melanosomes and neuron synaptic vesicles and has always been proposed as the candidate gene for the autosomal recessive Griscelli–Pruniéras Syndrome, one of the silvery hair Syndromes, which is a lethal disease combining immunodeficiency and neurologic and pigmentary abnormalities. Thus far, two mutations in the myosin Va gene have been described to be associated with this Syndrome. One of these mutations was a homozygous mis-sense mutation causing an arginine to cysteine alteration at codon 1246. Because we also found this particular substitution after mutation analysis of a Griscelli patient, we checked its relevance in a control group of 124 unrelated healthy individuals and found it to be present, even in homozygous state, in normal unaffected individuals. It is clear that this arg1246cys substitution is a polymorphism occurring in the human population and not occurring in association with Griscelli Syndrome. Distinguishing a polymorphism from a bona fide mutation is of uttermost importance and has major ethical implications with regard to prenatal genetic counseling in affected families
Wendy Westbroek - One of the best experts on this subject based on the ideXlab platform.
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novel 47 5 kb deletion in rab27a results in severe Griscelli Syndrome type 2
Molecular Genetics and Metabolism, 2010Co-Authors: Lisa M Vincent, Wendy Westbroek, Fred Gilbert, Jennifer Dipace, Carla Ciccone, Thomas C Markello, Andrew Jeong, Heidi Dorward, William A Gahl, James B BusselAbstract:Griscelli Syndrome (GS), a rare autosomal recessive disorder characterized by partial albinism and immunological impairment and/or severe neurological impairment, results from mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes. We identified a Hispanic patient born of a consanguineous union who presented with immunodeficiency, partial albinism, hepatic dysfunction, hemophagocytosis, neurological impairment, nystagmus, and silvery hair indicative of Griscelli Syndrome Type 2 (GS2). We screened for point mutations, but only exons 2-6 of the patient's DNA could be PCR-amplified. Whole genome analysis using the Illumina 1M-Duo DNA Analysis BeadChip identified a homozygous deletion in the patient's DNA. The exact breakpoints of the 47.5-kb deletion were identified as chr15q15-q21.1: g.53332432_53379990del (NCBI Build 37.1); the patient lacks the promoter and 5'UTR regions of RAB27A, thus confirming the diagnosis of GS2.
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A novel missense mutation (G43S) in the switch I region of Rab27A causing Griscelli Syndrome
Molecular genetics and metabolism, 2008Co-Authors: Wendy Westbroek, Maya Tuchman, Bradford Tinloy, Olivier De Wever, Thierry Vilboux, Jens Michael Hertz, Henrik Hasle, Carsten Heilmann, Amanda Helip-wooley, Robert KletaAbstract:The autosomal recessive Griscelli Syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking steps within cells. In melanocytes, the GTP-bound form of Rab27A associates with the membranes of mature fully-pigmented melanosomes through its geranylgeranyl group. Once attached, Rab27A recruits the downstream effector Melanophilin (Mlph) and the actin-dependent motor protein Myosin Va (MyoVa). The molecular Rab27A/Mlph/MyoVA tripartite complex, which links melanosomes to the peripheral actin network, is required to achieve melanosome transfer to surrounding keratinocytes in the epidermis. Here we report a novel homozygous missense mutation c.127G>A, p.G43S in exon 2 of the RAB27A gene of an Afghani GSII patient. Laser scanning confocal microscopy showed that the G43S mutation, which is located in the highly conserved switch I region of Rab27A, induces perinuclear localization of melanosomes in normal melanocytes, and fails to restore melanosomes to the actin-rich periphery in GSII melanocytes. Co-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins.
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Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli Syndrome
The Journal of biological chemistry, 2003Co-Authors: Philippe Bahadoran, Mitsunori Fukuda, Roser Buscà, Karine Bille, Christine Chiaverini, Wendy Westbroek, Jo Lambert, Gaëlle Valony, Jean-marie Naeyaert, Jean-paul OrtonneAbstract:Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli Syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.
C Griscelli - One of the best experts on this subject based on the ideXlab platform.
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partial albinism with immunodeficiency Griscelli Syndrome
The Journal of Pediatrics, 1994Co-Authors: Christoph Klein, Alain Fischer, Anne Durandy, Francoise Le Deist, N Philippe, S Fraitag, C Prost, C GriscelliAbstract:Abstract Partial albinism with immunodeficiency is a rare and fatal immunologic disorder characterized by pigmentary dilution and variable cellular immunodeficiency. To define the phenotype, therapy, and outcome, we retrospectively analyzed seven consecutive patients. Primary abnormalities included a silvery-grayish sheen to the hair, large pigment agglomerations in hair shafts, and an abundance of mature melanosomes in melanocytes, with reduced pigmentation of adjacent keratinocytes. Clinical onset occurred between the ages of 4 months and 4 years and was characterized by accelerated phases (lymphohistiocytic infiltration of multiple organs, including the brain and the meninges), triggered by viral and bacterial infections. Characteristic laboratory features included pancytopenia, hypofibrinogenemia, hypertriglyceridemia, and hypoproteinemia. Consistent immunologic abnormalities were characterized by absent delayed-type cutaneous hypersensitivity and impaired natural killer cell function. Some patients had secondary hypogammaglobulinemia, impaired major histocompatibility complex-mediated cytotoxic effects, a decreased capacity of lymphocytes to trigger a mixed lymphocyte reaction, or various functional granulocytic abnormalities. The disease seems to be invariably lethal without bone marrow transplantation; the mean age at the time of death was 5 years. Bone marrow transplantation has been performed in three cases; two patients died in the immediate posttransplantation period of infectious complications, but one patient is cured after a follow-up of 5 years. We conclude that partial albinism with immunodeficiency (Griscelli Syndrome) can be differentiated from Chediak-Higashi Syndrome by pathognomonic histologic features. One of the underlying immunologic defects may be a defective function of natural killer cells, predisposing the patient to virus-associated hemophagocytic Syndrome or accelerated phases. The prognosis is very poor unless early bone marrow transplantation is carried out. (J P EDIATR 1994;125:886-95)
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prenatal diagnosis of Syndromes associating albinism and immune deficiencies chediak higashi Syndrome and variant
Prenatal Diagnosis, 1993Co-Authors: Anne Durandy, J Bretongorius, Delphine Guygrand, C Dumez, C GriscelliAbstract:We have successfully undertaken the prenatal diagnosis of two hereditary Syndromes associating albinism and immune defects. Because the genes responsible for these diseases have not yet been mapped and the immune abnormalities are too subtle to be diagnosed in utero, the prenatal diagnosis was made using a morphological approach. In the case of Chediak-Higashi Syndrome, it was based on light microscopic examination of the hair shaft and on light and electron microscopic study of polymorphonuclear cells. In the Syndrome associating immune deficiency and partial albinism, the Griscelli Syndrome, only examination of the hair was feasible. The diagnosis was negative in 12 fetuses at risk and positive in four.
