Membranous complexes characteristic of melanocytes derived from patients with Hermansky–Pudlak syndrome type 1 are macroautophagosomal entities of the lysosomal compartment

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder resulting from mutations in a family of genes required for efficient transport of lysosomal‐related proteins from the trans‐Golgi network to a target organelle. To date, there are several genetically distinct forms of HPS. Many forms of HPS exhibit aberrant trafficking of melanosome‐targeted proteins resulting in incomplete melanosome biogenesis responsible for oculocutaneous albinism observed in patients. In HPS‐1, melanosome‐targeted proteins are localized to characteristic membranous complexes, which have morphologic similarities to macroautophagosomes. In this report, we evaluated the hypothesis that HPS‐1‐specific membranous complexes comprise a component of the lysosomal compartment of melanocytes. Using indirect immunofluorescence, an increase in co‐localization of misrouted tyrosinase with cathepsin‐L, a lysosomal cysteine protease, occurred in HPS‐1 melanocytes. In addition, ribophorin II, an integral endoplasmic reticulum protein that is also a component of macroautophagosomes, and LC3, a specific marker of macrophagosomes, demonstrated localization to membranous complexes in HPS‐1 melanocytes. At the electron microscopic level, the membranous complexes exhibited acid phosphatase activity and localization of exogenously supplied horseradish peroxidase (HRP)‐conjugated gold particles, indicating incorporation of lysosomal and endosomal components to membranous complexes, respectively. These results confirm that membranous complexes of HPS‐1 melanocytes are macroautophagosomal representatives of the lysosomal compartment.     

BLOC-3, a protein complex containing the Hermansky-Pudlak syndrome gene products HPS1 and HPS4

The Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. HPS results from mutations in either one of six human genes named HPS1 to HPS6, most of which encode proteins of unknown function. Here we report that the human HPS1 and HPS4 proteins are part of a complex named BLOC-3 (for biogenesis of lysosome-related organelles complex 3). Co-immunoprecipitation experiments demonstrated that epitope-tagged and endogenous HPS1 and HPS4 proteins assemble with each other in vivo. The HPS1.HPS4 complex is predominantly cytosolic, with a small amount being peripherally associated with membranes. Size exclusion chromatography and sedimentation velocity analyses of the cytosolic fraction indicate that HPS1 and HPS4 form a moderately asymmetric protein complex with a molecular mass of approximately 175 kDa. HPS4-deficient fibroblasts from light ear mice display normal distribution and trafficking of the lysosomal membrane protein, Lamp-2, in contrast to fibroblasts from AP-3-deficient pearl mice (HPS2), which exhibit increased trafficking of this lysosomal protein via the plasma membrane. Similarly, light ear fibroblasts display an apparently normal accumulation of Zn2+ in intracellular vesicles, unlike pearl fibroblasts, which exhibit a decreased intracellular Zn2+ storage. Taken together, these observations demonstrate that the HPS1 and HPS4 proteins are components of a cytosolic complex that is involved in the biogenesis of lysosomal-related organelles by a mechanism distinct from that operated by AP-3 complex.     

Membranous complexes characteristic of melanocytes derived from patients with Hermansky-Pudlak syndrome type 1 are macroautophagosomal entities of the lysosomal compartment

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder resulting from mutations in a family of genes required for efficient transport of lysosomal-related proteins from the trans-Golgi network to a target organelle. To date, there are several genetically distinct forms of HPS. Many forms of HPS exhibit aberrant trafficking of melanosome-targeted proteins resulting in incomplete melanosome biogenesis responsible for oculocutaneous albinism observed in patients. In HPS-1, melanosome-targeted proteins are localized to characteristic membranous complexes, which have morphologic similarities to macroautophagosomes. In this report, we evaluated the hypothesis that HPS-1-specific membranous complexes comprise a component of the lysosomal compartment of melanocytes. Using indirect immunofluorescence, an increase in co-localization of misrouted tyrosinase with cathepsin-L, a lysosomal cysteine protease, occurred in HPS-1 melanocytes. In addition, ribophorin II, an integral endoplasmic reticulum protein that is also a component of macroautophagosomes, and LC3, a specific marker of macrophagosomes, demonstrated localization to membranous complexes in HPS-1 melanocytes. At the electron microscopic level, the membranous complexes exhibited acid phosphatase activity and localization of exogenously supplied horseradish peroxidase (HRP)-conjugated gold particles, indicating incorporation of lysosomal and endosomal components to membranous complexes, respectively. These results confirm that membranous complexes of HPS-1 melanocytes are macroautophagosomal representatives of the lysosomal compartment.     

Molecular characterization of the protein encoded by the Hermansky-Pudlak syndrome type 1 gene

Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of approximately 80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was approximately 6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the beta3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1 gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex.     

Hermansky–Pudlak Syndrome and Pale Ear: Melanosome‐Making for the Millennium

Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid‐lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles – melanosomes, platelet granules, and lysosomes – suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high‐molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.     

Clinico‐molecular analysis of eleven patients with Hermansky–Pudlak type 5 syndrome,a mild form of HPS

Hermansky–Pudlak syndrome (HPS), first described in 1959, is a rare form of syndromic oculocutaneous albinism associated with bleeding diathesis and in some cases pulmonary fibrosis and granulomatous colitis. All 10 HPS types are caused by defects in vesicle trafficking of lysosome‐related organelles (LRO) proteins. The HPS5 protein associates with HPS3 and HPS6 to form the biogenesis of lysosome‐related organelles complex‐2 (BLOC‐2). Here, we report the clinical and genetic data of 11 patients with HPS‐5 analyzed in our laboratory. We report 11 new pathogenic variants. The 11 patients present with ocular features that are typical for albinism, with mild hypopigmentation, and with no other major complication, apart from a tendency to bleed. HPS‐5 therefore appears as a mild form of HPS, which is often clinically undistinguishable from mild oculocutaneous or ocular forms of albinism. Molecular analysis is therefore required to establish the diagnosis of this mild HPS form, which has consequences in terms of prognosis and of clinical management of the patients.     

A BLOC-1 mutation screen reveals a novel BLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1-6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient's melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.     

Characterization of melanosomes and melanin in Japanese patients with Hermansky–Pudlak syndrome types 1, 4, 6, and 9

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism (OCA), a bleeding tendency, and ceroid deposition. Most of the causative genes for HPS encode subunits of the biogenesis of lysosome‐related organelles complex (BLOC). In this study, we identified one patient each with HPS4, HPS6, and HPS9 by whole‐exome sequencing. Next, we analyzed hair samples from the three patients and representative patients with HPS1 and controls using electron microscopy and chemical methods. All HPS patients had fewer, smaller, and more immature melanosomes than healthy controls. Further, all patients showed reduced total melanin content and increased levels of benzothiazine‐type pheomelanin. The results of this study demonstrate the impact of the dysfunctions of BLOCs on the maturation of melanosomes and melanin levels and composition through analysis of their hair samples.     

Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes

Hermansky-Pudlak syndrome (HPS) has evolved into a group of genetically distinct disorders characterized by oculocutaneous albinism, a storage pool deficiency, and impaired formation or trafficking of intracellular vesicles. HPS-1 results from mutations in the HPS1 gene and affects approximately 400 individuals in northwest Puerto Rico due to a 16-bp duplication in exon 15. Another 13 mutations have been reported in non-Puerto Ricans. HPS1 codes for a 79.3 kDa cytoplasmic protein of unknown function. HPS-1 patients typically develop fatal pulmonary fibrosis in their fourth decade. HPS-2 is caused by mutations in ADTB3A, which codes for the beta3A subunit of the adaptor protein-3 complex, AP3. This coat protein complex has been localized to the TGN as well as to a peripheral endosomal compartment. Evidence indicates that AP3 plays a role in the stepwise process of vesicular trafficking which leads to formation of the melanosomal, platelet dense body and lysosomal compartments. All three known HPS-2 patients had childhood neutropenia and infections. HPS-3 results from mutations in HPS3 and affects central Puerto Ricans homozygous for a 3904-bp deletion removing exon 1. At least 8 non-Puerto Rican patients have other HPS3 mutations, including an IVS5+1G->A splicing mutation in five Ashkenazi Jewish patients. HPS3 codes for a 113.7 kDa protein of unknown function. HPS-3 manifests with mild hypopigmentation and bleeding. All types of HPS are diagnosed by whole mount electron microscopic demonstration of absent platelet dense bodies, and molecular diagnoses are available for the Puerto Rican HPS1 and HPS3 founder mutations. Mouse and Drosophila models provide candidates for new genes causing HPS in humans. These genes will reveal the pathways by which specialized vesicles of lysosomal lineage arise within cells.     

The Hermansky-Pudlak syndrome 1 (HPS1) and HPS4 proteins are components of two complexes,BLOC-3 and BLOC-4, involved in the biogenesis of lysosome-related organelles

Hermansky-Pudlak syndrome (HPS) is a genetic disease of lysosome, melanosome, and granule biogenesis. Mutations of six different loci have been associated with HPS in humans, the most frequent of which are mutations of the HPS1 and HPS4 genes. Here, we show that the HPS1 and HPS4 proteins are components of two novel protein complexes involved in biogenesis of melanosome and lysosome-related organelles: biogenesis of lysosome-related organelles complex-(BLOC) 3 and BLOC-4. The phenotypes of Hps1-mutant (pale-ear; ep) and Hps4-mutant (light-ear; le) mice and humans are very similar, and cells from ep and le mice exhibit similar abnormalities of melanosome morphology. HPS1 protein is absent from ep-mutant cells, and HPS4 from le-mutant cells, but le-mutant cells also lack HPS1 protein. HPS4 protein seems to be necessary for stabilization of HPS1, and the HPS1 and HPS4 proteins co-immunoprecipitate, indicating that they are in a complex. HPS1 and HPS4 do not interact directly in a yeast two-hybrid system, although HPS4 interacts with itself. In a partially purified vesicular/organellar fraction, HPS1 and HPS4 are both components of a complex with a molecular mass of approximately 500 kDa, termed BLOC-3. Within BLOC-3, HPS1 and HPS4 are components of a discrete approximately 200-kDa module termed BLOC-4. In the cytosol, HPS1 (but not HPS4) is part of yet another complex, termed BLOC-5. We propose that the BLOC-3 and BLOC-4 HPS1.HPS4 complexes play a central role in trafficking cargo proteins to newly formed cytoplasmic organelles.     

Hermansky–Pudlak Syndrome: Vesicle Formation from Yeast to Man

The disorders known as Hermansky–Pudlak syndrome (HPS) are a group of genetic diseases resulting from abnormal formation of intracellular vesicles. In HPS, dysfunction of melanosomes results in oculocutaneous albinism, and absence of platelet dense bodies causes a bleeding diathesis. In addition, some HPS patients suffer granulomatous colitis or fatal pulmonary fibrosis, perhaps due to mistrafficking of a subset of lysosomes. The impaired function of specific organelles indicates that the causative genes encode proteins operative in the formation of certain vesicles. Four such genes, HPS1, ADTB3A, HPS3, and HPS4, are associated with the four known subtypes of HPS, i.e. HPS‐1, HPS‐2, HPS‐3, and HPS‐4. ADTB3A codes for the β3A subunit of adaptor complex‐3, known to assist in vesicle formation from the trans‐Golgi network or late endosome. However, the functions of the HPS1, HPS3, and HPS4 gene products remain unknown. These three genes arose with the evolution of mammals and have no homologs in yeast, reflecting their specialized function. In contrast, all four known HPS‐causing genes have homologs in mice, a species with 14 different models of HPS, i.e. hypopigmentation and a platelet storage pool deficiency. Pursuit of the mechanism of mammalian vesicle formation and trafficking, impaired in HPS, relies upon investigation of these mouse models as well as studies of protein complexes involved in yeast vacuole formation.     

The Hermansky-Pudlak syndrome 3 (cocoa) protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2)

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease affecting vesicle trafficking among lysosome-related organelles. The Hps3, Hps5, and Hps6 genes are mutated in the cocoa, ruby-eye-2, and ruby-eye mouse pigment mutants, respectively, and their human orthologs are mutated in HPS3, HPS5, and HPS6 patients. These three genes encode novel proteins of unknown function. The phenotypes of Hps5/Hps5,Hps6/Hps6 and Hps3/Hps3,Hps6/Hps6 double mutant mice mimic, in coat and eye colors, in melanosome ultrastructure, and in levels of platelet dense granule serotonin, the corresponding phenotypes of single mutants. These facts suggest that the proteins encoded by these genes act within the same pathway or protein complex in vivo to regulate vesicle trafficking. Further, the Hps5 protein is destabilized within tissues of Hps3 and Hps6 mutants, as is the Hps6 protein within tissues of Hps3 and Hps5 mutants. Also, proteins encoded by these genes co-immunoprecipitate and occur in a complex of 350 kDa as determined by sucrose gradient and gel filtration analyses. Together, these results indicate that the Hps3, Hps5, and Hps6 proteins regulate vesicle trafficking to lysosome-related organelles at the physiological level as components of the BLOC-2 (biogenesis of lysosome-related organelles complex-2) protein complex and suggest that the pathogenesis and future therapies of HPS3, HPS5, and HPS6 patients are likely to be similar. Interaction of the Hps5 and Hps6 proteins within BLOC-2 is abolished by the three-amino acid deletion in the Hps6(ru) mutant allele, indicating that these three amino acids are important for normal BLOC-2 complex formation.     

The cell biology of Hermansky-Pudlak syndrome: recent advances

Hermansky-Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding. These manifestations arise from defects in the biogenesis of lysosome-related organelles, including melanosomes and platelet dense granules. Most genes associated with HPS in humans and rodent models of the disease encode components of multisubunit protein complexes that are expressed ubiquitously and play roles in intracellular protein trafficking and/or organelle distribution. A small GTPase of the Rab family, Rab38, is also implicated in the pathogenesis of the disease. This article reviews recent progress toward elucidating the cellular functions of these proteins.     

Hermansky-Pudlak syndrome and pale ear: melanosome-making for the millennium

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid-lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles--melanosomes, platelet granules, and lysosomes--suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high-molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.     

BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.     

Instability of BLOC‐2 and BLOC‐3 in Chinese patients with Hermansky‐Pudlak syndrome

Hermansky‐Pudlak syndrome (HPS) is a rare recessive disorder characterized by oculocutaneous albinism (OCA) or ocular albinism (OA), bleeding tendency, and other symptoms due to multiple defects in tissue‐specific lysosome‐related organelles. Ten HPS subtypes have been characterized with mutations in HPS1 to HPS10, which encode the subunits of BLOC‐1, ‐2, ‐3, and AP‐3. Using next‐generation sequencing (NGS), we have screened 100 hypopigmentation genes in OCA or OA patients and identified four HPS‐1, one HPS‐3, one HPS‐4, one HPS‐5, and three HPS‐6. The HPS‐4 case is the first report in the Chinese population. Among these 20 mutational alleles, 16 were previously unreported alleles (6 in HPS1, 1 in HPS3, 2 in HPS4, 2 in HPS5, and 5 in HPS6). BLOC‐2 and BLOC‐3 were destabilized due to the mutation of these HPS genes which are so far the only reported causative genes in Chinese HPS patients, in which HPS‐1 and HPS‐6 are the most common subtypes. The mutational spectrum of Chinese HPS is population specific.     

Biogenesis of Lysosome-related Organelles Complex-1 Subunit 1 (BLOS1) Interacts with Sorting Nexin 2 and the Endosomal Sorting Complex Required for Transport-I (ESCRT-I) Component TSG101 to Mediate the Sorting of Epidermal Growth Factor Receptor into Endosomal Compartments

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a component of the molecular machinery required for the biogenesis of specialized organelles and lysosomal targeting of cargoes via the endosomal to lysosomal trafficking pathway. BLOS1, one subunit of BLOC-1, is implicated in lysosomal trafficking of membrane proteins. We found that the degradation and trafficking of epidermal growth factor receptor (EGFR) were delayed in BLOS1 knockdown cells, which were rescued through BLOS1 overexpression. A key feature to the delayed EGFR degradation is the accumulation of endolysosomes in BLOS1 knockdown cells or BLOS1 knock-out mouse embryonic fibroblasts. BLOS1 interacted with SNX2 (a retromer subunit) and TSG101 (an endosomal sorting complex required for transport subunit-I) to mediate EGFR lysosomal trafficking. These results suggest that coordination of the endolysosomal trafficking proteins is important for proper targeting of EGFR to lysosomes.     

Hermansky–Pudlak syndrome: a disease of protein trafficking and organelle function

The Hermansky–Pudlak syndrome (HPS) is a collection of related autosomal recessive disorders which are genetically heterogeneous. There are eight human HPS subtypes, characterized by oculocutaneous albinism and platelet storage disease; prolonged bleeding, congenital neutropenia, pulmonary fibrosis, and granulomatous colitis can also occur. HPS is caused primarily by defects in intracellular protein trafficking that result in the dysfunction of intracellular organelles known as lysosome‐related organelles. HPS gene products are all ubiquitously expressed and all associate in various multi‐protein complexes, yet HPS has cell type‐specific disease expression. Impairment of specialized secretory cells such as melanocytes, platelets, lung alveolar type II epithelial cells and cytotoxic T cells are observed in HPS. This review summarizes recent molecular, biochemical and cell biological analyses together with clinical studies that have led to the correlation of molecular pathology with clinical manifestations and led to insights into such diverse disease processes such as albinism, fibrosis, hemorrhage, and congenital neutropenia.     

Murine Hermansky-Pudlak syndrome genes: regulators of lysosome-related organelles

In the mouse, at least 16 genes regulate vesicle trafficking to specialized lysosome-related organelles, including platelet dense granules and melanosomes. Fourteen of these genes have been identified by positional cloning. All 16 mouse mutants are models for the genetically heterogeneous human disease, Hermansky-Pudlak Syndrome (HPS). Five HPS genes encode known vesicle trafficking proteins. Nine genes are novel, are found only in higher eukaryotes and encode members of three protein complexes termed BLOCs (Biogenesis of Lysosome-related Organelles Complexes). Mutations in murine HPS genes, which encode protein co-members of BLOCs, produce essentially identical phenotypes. In addition to their well-known effects on pigmentation, platelet function and lysosome secretion, HPS genes control a wide range of physiological processes including immune recognition, neuronal functions and lung surfactant trafficking. Studies of the molecular functions of HPS proteins will reveal important details of vesicle trafficking and may lead to therapies for HPS.