Melanosome Morphologies in Murine Models of Hermansky–Pudlak Syndrome Reflect Blocks in Organelle Development

Nguyen, Thuyen; Novak, Edward K.; Kermani, Maryam; Fluhr, Joachim W.; Peters, Luanne L.; Swank, Richard T.; Wei, Maria L.

doi:10.1046/j.1523-1747.2002.19535.x

Cited by 72 publications

(93 citation statements)

References 43 publications

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“…We found that mutations in DTNBP1/Dtnbp1 cause HPS-7/sdy, providing the first evidence to our knowledge that a mutation affecting a BLOC-1 complex component causes HPS in humans. The phenotypes of the four BLOC-1 mutants (pallid, muted, cappuccino and sandy) are the most severe of the mouse HPS mutants 6,22 , emphasizing the importance of BLOC-1 in the biogenesis of lysosome-related organelles, and their similar phenotypes 6,22 are consistent with their encoded proteins forming a common complex. One interpretation of the fact that dystrobrevin interacts with components (dysbindin and muted) of the BLOC-1 complex is that DPC components are involved in vesicle trafficking in nonmuscle tissues.…”

mentioning

confidence: 81%

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

et al. 2003

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Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules [1][2][3] . In mice, at least 16 loci are associated with HPS 4-6 , including sandy (sdy ; ref. 7 ). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to α-and β-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells 8 . We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 ), which regulates Correspondence should be addressed to R.T.S (richard.swank@roswellpark.org). 11 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Genetics website. Competing Interests Statement:The authors declare that they have no competing financial interests. We previously showed 7 that the sdy mutant mouse is a valid model for human HPS and localized the gene sdy to mouse chromosome 13. Here we genotyped 20 microsatellite markers in 1,250 progeny of sdy backcrosses to localize sdy to the 2.2-cM interval between D13Mit244 and D13Mit267 (Fig. 1). We identified the sdy interval within a 28-Mb scaffold (Celera Discovery System) containing two known genes, Jmj and Dtnbp1 (Fig. 1b). We used PCR products of D13Mit179 and the Dtnbp1 cDNA as probes to generate a BAC contig covering the sdy interval (Fig. 1b). NIH Public AccessNorthern-blot analysis and sequencing of RT-PCR products of Jmj identified no abnormalities in sdy mutants, but truncated genomic PCR products (Fig. 2a) and mRNA ( Fig. 2b) of Dtnbp1 were apparent in sdy tissues. Sequencing of RT-PCR products showed that exons 6 and 7 (156 bp) of Dtnbp1 were deleted in mutant mice, resulting in the loss of 52 amino acids from position 119-172 of the dysbindin protein ( Supplementary Fig. 1 online). Genomic sequencing showed that this results from a large deletion (38,129 bp) from nucleotide 3,701 of intron 5 to nucleotide 12,377 of intron 7. This deletion was not found in twelve other inbred mouse strains (Fig. 2a), including coisogenic DBA/2J, indicating that it was not a strain-specific polymorphism. This in-frame deletion creates a 1.5-kb mutant dysbindin transcript ( Fig. 2b) and abolishes expression of the 51-kDa dysbindin 8 protein in sdy/sdy mice ( Fig. 2c). Expression of dysbindin is restored in sdy/sdy transgenic mice containing BAC54F9 (Fig. 2c). Platelet serotonin levels of six of these transgenics were normal (>1.12 μg per 10 9 platelets), whereas all five sdy/sdy litter-mates without BAC54F9 had platelet serotonin levels of <0.06 μg per 10 9 platelets. sdy/sdy progeny containing the BAC transgene had dar...

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confidence: 81%

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

et al. 2003

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“…Samples were prepared (with minor modifications as noted in the SI Materials and Methods), and the percentage of melanosomes was determined as previously published (14). At least 10 individual melanoma cells were evaluated for each cell line or condition.…”

Section: Methodsmentioning

confidence: 99%

“…The Hps3 protein is part of a multimeric complex, BLOC-2, that regulates trafficking from the endosome; BLOC-2 also includes the subunit Hps5 and Hps6 proteins (22). We previously demonstrated that mutation of Hps3, Hps5, or Hps6 genes resulted in aberrant melanosome formation (14). To test if alteration of the function of BLOC-2 could influence chemosensitivity, we examined the effects of mutation of Hps6 on treatment sensitivity to cDDP.…”

Section: Chemosensitivity Is Up-regulated or Down-regulated By Receptmentioning

confidence: 99%

“…Mutations in VPS33A and CNO cause cell type-specific symptoms in patients and mice largely limited to melanocytes and platelets resulting in pigment dilution and nonlife-threatening prolonged bleeding times (8). Mutations in the Vps33a or Cno gene both resulted in increased sensitivity to cDDP, etoposide, and vinblastine (13) and blocked the normal process of eumelanosome maturation through four morphological stages (stages I-IV) (10,14), indicating a fundamental role for VPS33A and CNO in contributing to cellular resistance to multiple cytotoxic agents.…”

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confidence: 99%

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Targeting protein-trafficking pathways alters melanoma treatment sensitivity

Huang

Chinen

Chang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the proteintrafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocytestimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.

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“…Cappuccino, pallidin, and dysbindin were originally identified as proteins that cause a severe pigmentation defect in mice when mutated 12,13 . These three proteins are components of the biogenesis of lysosome-related organelles complex 1 (BLOC-1), required for the proper sorting of cargo (such as TYRP1) from early endosomes to lysosome-related organelles 14 .…”

Section: Introductionmentioning

confidence: 99%

Technical Note: The Use of RNA-interference as a Tool to Find Proteins Involved in Melanosome Formation or Transport

Amsen

Rotin

2009

Nature Precedings

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Abstract:Melanosomes are lysosome-related organelles that produce and transport the pigment melanin within melanocytes. Mutations in proteins required for melanosome transport and formation lead to a range of pigmentation defects, manifested at the cellular level as perinuclear clustering of melanosomes, or reduced sorting of melanosomal cargo such as tyrosinase-related protein 1 (TYRP1). A pilot screen was carried out to investigate whether a combination of cellular imaging and RNA interference could be used to identify new proteins involved in pigmentation pathways. In this study, eleven genes known to play a role in melanosome transport/formation or other pigmentation properties were knocked down in mouse melanocytes with shRNAmir constructs. The investigated genes were TYRP1, pallidin, cappuccino, dysbindin, HPS5, LYST, Myosin Va, melanophilin, RhoA, UBPY and mahogunin. In a blinded confocal imaging experiment, the only reproducible change observed in cells in which these targets were knocked down was a decrease in TYRP1 levels upon transfection with knockdown constructs against TYRP1 itself, or one of three constructs targeting HPS5 (Hermansky-Pudlak Syndrome 5). Upon analysis with high-content imaging software, only the knockdown construct against TYRP1 itself was detected. RT-PCR analysis showed that many of the shRNAmir constructs did not reduce mRNA and proteins levels enough to detect effects on melanosome properties. This was further examined for melanophilin, a protein necessary for melanosome transport. Altogether, the data show that this system is currently not sensitive enough for use in a screen for unknown regulators of melanosome transport and formation. The main obstacle appears to be incomplete reduction of target protein levels. Our observation that a ~50% reduction in mRNA level is not sufficient to elicit an effect is supported by the fact that heterozygous carriers of melanosomal transport disorders (Griscelli Syndrome, Hermansky-Pudlak Syndrome) do not display diseases phenotypes. A further reduction in protein levels, for example by viral integration of shRNA, may be required.

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Melanosome Morphologies in Murine Models of Hermansky–Pudlak Syndrome Reflect Blocks in Organelle Development

Cited by 72 publications

References 43 publications

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

Targeting protein-trafficking pathways alters melanoma treatment sensitivity

Technical Note: The Use of RNA-interference as a Tool to Find Proteins Involved in Melanosome Formation or Transport

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