A homozygous AP3D1 missense variant in patients with sensorineural hearing loss as the leading manifestation.

Loss-of-function variants in AP3D1 have been linked to Hermansky-Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein-protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

Connecting COPD GWAS Genes: FAM13A Controls TGFß2 Secretion by Modulating AP-3 Transport.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein-protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)-AP3D1-CTGF- TGFß2. We find that TGFß2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFß2 through an AP-3 (adaptor protein 3)-dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat-cargo interaction. Moreover, we find that TGFß2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.

Mutations in AP3D1 associated with immunodeficiency and seizures define a new type of Hermansky-Pudlak syndrome.

Genetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3ß3A subunit, affected in HPS2 patients, is substituted by AP3ß3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.

Defective HIV-1 particle assembly in AP-3-deficient cells derived from patients with Hermansky-Pudlak syndrome type 2.

Adaptor protein complex 3 (AP-3) is a heterotetramer that is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 deficiency in humans, induced by mutations in the AP3B1 gene, which encodes the ß3A subunit of the AP-3 complex, results in Hermansky-Pudlak syndrome 2 (HPS2), which is a rare genetic disorder with defective lysosome-related organelles. In a previous study, we identified the AP-3 complex as an important contributor to HIV-1 assembly and release. We hypothesized that cells from patients affected by HPS2 should demonstrate abnormalities of HIV-1 assembly. Here we report that HIV-1 particle assembly and release are indeed diminished in HPS2 fibroblast cultures. Transient or stable expression of the full-length wild-type ß3A subunit in HPS2 fibroblasts restored the impaired virus assembly and release. In contrast, virus-like particle release mediated by MA-deficient Gag mutants lacking the AP-3 binding site was not altered in HPS2 cells, indicating that the MA domain serves as the major viral determinant required for the recruitment of the AP-3 complex. AP-3 deficiency decreased HIV-1 Gag localization at the plasma membrane and late endosomes and increased the accumulation of HIV-1 Gag at an intermediate step between early and late endosomes. Blockage of the clathrin-mediated endocytic pathway in HPS2 cells did not reverse the inhibited virus assembly and release imposed by the AP-3 deficiency. These results demonstrate that the intact and stable AP-3 complex is required for HIV-1 assembly and release, and the involvement of the AP-3 complex in late stages of the HIV-1 replication cycle is independent of clathrin-mediated endocytosis.

Serum anti-AP3D1 antibodies are risk factors for acute ischemic stroke related with atherosclerosis.

Atherosclerosis has been considered as the main cause of morbidity, mortality, and disability worldwide. The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning, which has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen. It was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke (AIS), transient ischemic attack, diabetes mellitus (DM), cardiovascular disease, chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were higher than those of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The results of the Japan Public Health Center-based Prospective Study indicated that this antibody marker is deemed useful as risk factors for AIS.

A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers.

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.

Deletion mutants of AP-1 adaptin subunits display distinct phenotypes in fission yeast.

Adaptins are subunits of the heterotetrameric (beta/mu/gamma/sigma) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the Deltaapm1 and Deltaapl2 mutants displayed similar but more severe phenotypes than those of Deltaaps1 or Deltaapl4 mutants. Furthermore, the Deltaapl2Deltaaps1 and Deltaapl2Deltaapl4 double mutants displayed synthetic growth defects, whereas the Deltaaps1Deltaapl4 and Deltaapl2Deltaapm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes.

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex.

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

Biallelic mutations in AP3D1 cause Hermansky-Pudlak syndrome type 10 associated with immunodeficiency and seizure disorder.

Several types of Hermansky-Pudlak syndromes (HPS) represent a group of immunodeficiency syndromes that feature both leukocyte defects with partial albinism of hair, skin, and eyes. These conditions share defects in genes that encode proteins involved in the biogenesis, function, and trafficking of secretory lysosomes. Mutations in AP3D1 which encode the main subunit AP-3(δ) were recently reported on one individual and led to Hermansky-Pudlak Syndrome type 10 (HPS10; OMIM 617050). HPS10 is a severe condition that manifests with symptoms of oculocutaneous albinism, neurodevelopmental delays, platelet dysfunction, and immunodeficiency. Herein we report on three affected individuals who presented with severe seizures, developmental delay, albinism, and immunodeficiency. Whole exome sequencing identified homozygosity for a deleterious sequence variant of high impact in AP3D1, c.1978delG, predicting p.Ala660Argfs*54 (NM_001261826.3). We further demonstrated an abnormal storage pathway in the platelets. The current study represents a second confirmation report and implicates AP3D1 mutations as a cause of Hermansky-Pudlak Syndrome type 10.

In silico and in vitro analysis of boAP3d1 protein interaction with bovine leukaemia virus gp51.

The envelope glycoprotein 51 (gp51) is essential for bovine leukaemia virus (BLV) entry to bovine B-lymphocytes. Although the bovine adaptor protein 3 complex subunit delta-1 (boAP3D1) has been proposed as the potential receptor, the specific ligand-receptor interaction has not yet been completely defined and boAP3D1 receptor and gp51 3D structures have not been determined. This study was thus aimed at a functional annotation of boAP3D1 cellular adaptor protein and BLV gp51 and, proposing a reliable model for gp51-AP3D1 interaction using bioinformatics tools. The boAP3D1 receptor interaction patterns were calculated based on models of boAP3D1 receptor and gp51 complexes' 3D structures, which were constructed using homology techniques and data-driven docking strategy. The results showed that the participation of 6 key amino acids (aa) on gp51 (Asn170, Trp127, His115, Ala97, Ser98 and Glu128) and 4 aa on AP3D1 (Lys925, Asp807, Asp695 and Arg800) was highly probable in the interaction between gp51 and BLVR domains. Three gp51 recombinant peptides were expressed and purified to validate these results: the complete domain (rgp51), the N-terminal portion (rNgp51) and the C-terminal fragment (rCgp51); and binding assays to Madin-Darby bovine kidney (MDBK) cells were then carried out with each recombinant. It was found that rNgp51 preferentially bound to MDBK cells, suggesting this domain's functional role during invasion. The rNgp51-MDBK cell interaction was sensitive to trypsin (98% reduction) and chymotrypsin treatment (80% reduction). These results highlighted that the N-terminal portion of gp51 interacted in vitro with the AP3D1 receptor and provides a plausible in silico interaction model.

Clathrin and adaptors.

Clathrin and adaptors are components of clathrin-coated pits and vesicles. The AP-1 adaptor complex is associated with clathrin-coated vesicles budding from the TGN, while the AP-2 adaptor complex is associated with clathrin-coated vesicles budding from the plasma membrane. The clathrin forms a polyhedral lattice and is believed to be the driving force behind membrane invagination leading to vesicle budding. The adaptors attach the clathrin to the membrane and also interact with the cytoplasmic domains of selected transmembrane proteins, causing these proteins to become concentrated in clathrin-coated vesicles. Clathrin-coated vesicles budding from the TGN have been implicated in the sorting of newly synthesised lysosomal enzymes, while clathrin-coated vesicles budding from the plasma membrane facilitate the receptor-mediated endocytosis of ligands, such as low density lipoproteins and transferrin. A novel adaptor-related complex, AP-3, has recently been identified, which is recruited onto membranes of the TGN and a more peripheral compartment but does not appear to be associated with clathrin. Genetic studies indicate that AP-3 plays a role in the sorting of proteins to lysosomes and lysosome-related organelles.

The AP-3 clathrin-associated complex is essential for embryonic and larval development in Caenorhabditis elegans.

The adaptor protein (AP) complexes are involved in membrane transport of many proteins. There are 3 AP complexes in C. elegans unlike mammals that have four. To study the biological functions of the AP-3 complexes of C. elegans, we sought homologues of the mouse and human genes that encode subunits of the AP-3 complexes by screening C. elegans genomic and EST sequences. We identified single copies of homologues of the m3, s3, b3 and d genes. The medium chain of AP-3 is encoded by a single gene in C. elegans but two different genes in mammals. Since there are no known mutations in these genes in C. elegans, we performed RNAi to assess their functions in development. RNAi of each of the genes caused embryonic and larval lethal phenotypes. APM-3 is expressed in most cells, particularly strongly in spermatheca and vulva. We conclude that the products of the C. elegans m3, s3, b3 and d genes are essential for embryogenesis and larval development.

A genome-wide association study identifies PLCL2 and AP3D1-DOT1L-SF3A2 as new susceptibility loci for myocardial infarction in Japanese.

Despite considerable progress in preventive and therapeutic strategies, myocardial infarction (MI) is one of the leading causes of death throughout the world. A total of 55 susceptibility genes have been identified mostly in European genome-wide association studies (GWAS). Nevertheless, large-scale GWAS from other population could possibly find additional susceptibility loci. To identify as many MI susceptibility loci as possible, we performed a large-scale genomic analysis in Japanese population. To identify MI susceptibility loci in Japanese, we conducted a GWAS using 1666 cases and 3198 controls using the Illumina Human610-Quad BeadChip and HumanHap550v3 Genotyping BeadChip. We performed replication studies using a total of 11,412 cases and 28,397 controls in the Japanese population. Our study identified two novel susceptibility loci for MI: PLCL2 on chromosome 3p24.3 (rs4618210:A>G, P = 2.60 × 10(-9), odds ratio (OR) = 0.91) and AP3D1-DOT1L-SF3A2 on chromosome 19p13.3 (rs3803915:A>C, P = 3.84 × 10(-9), OR = 0.89). Besides, a total of 14 previously reported MI susceptibility loci were replicated in our study. In particular, we validated a strong association on chromosome 12q24 (rs3782886:A>G: P = 1.14 × 10(-14), OR = 1.46). Following pathway analysis using 265 genes related to MI or coronary artery disease, we found that these loci might be involved in the pathogenesis of MI via the promotion of atherosclerosis. In the present large-scale genomic analysis, we identified PLCL2 and AP3D1-DOT1L-SF3A2 as new susceptibility loci for MI in the Japanese population. Our findings will add novel findings for MI susceptibility loci.

Novel cellular interacting partners of the human papillomavirus 16 transcription/replication factor E2.

Human papillomaviruses (HPVs) are causative agents in a number of human diseases. HPV can be divided into two groups: low risk that cause diseases such as genital warts, and high risk that cause ano-genital cancers. Of the high-risk group, HPV16 is the most commonly found in cervical cancer. All HPV encode an E2 protein and this protein regulates transcription from, and replication of, the viral genome making it essential for the viral life cycle. In order to function E2 must interact with cellular proteins; identification of these cellular partners will provide targets for disruption of the viral life cycle and will also provide insights into the processes of transcription and replication. To identify the cellular interacting partners for HPV16 E2, we carried out a yeast two-hybrid screen with the amino-terminus of E2 that is essential for mediating transcription and replication. Here we describe how this screen was carried out and detail the interacting partners that were identified; these include the proteins TopBP1, RACK1, POMP, p27(BBP), ODC antizyme, and Delta-adaptin. Several of these partners have characteristics that make them ideal candidates for mediating E2 function.

Fine-scale heterogeneity in crossover rate in the garnet-scalloped region of the Drosophila melanogaster X chromosome.

Homologous recombination affects myriad aspects of genome evolution, from standing levels of nucleotide diversity to the efficacy of natural selection. Rates of crossing over show marked variability at all scales surveyed, including species-, population-, and individual-level differences. Even within genomes, crossovers are nonrandomly distributed in a wide diversity of taxa. Although intra- and intergenomic heterogeneities in crossover distribution have been documented in Drosophila, the scale and degree of crossover rate heterogeneity remain unclear. In addition, the genetic features mediating this heterogeneity are unknown. Here we quantify fine-scale heterogeneity in crossover distribution in a 2.1-Mb region of the Drosophila melanogaster X chromosome by localizing crossover breakpoints in 2500 individuals, each containing a single crossover in this specific X chromosome region. We show 90-fold variation in rates of crossing over at a 5-kb scale, place this variation in the context of several aspects of genome evolution, and identify several genetic features associated with crossover rates. Our results shed new light on the scale and magnitude of crossover rate heterogeneity in D. melanogaster and highlight potential features mediating this heterogeneity.

Studying recombination with high-throughput sequencing: an educational primer for use with "fine-scale heterogeneity in crossover rate in the garnet-scalloped region of the Drosophila melanogaster X chromosome".

An article by Singh and colleagues in this issue of GENETICS quantifies variation in recombination rate across a small region of the Drosophila melanogaster genome, providing an opportunity for instructors of genetics to introduce or reinforce important concepts such as recombination and recombination rate variation, genome sequencing, and sequence features of the genome. Additional background information, a detailed explanation of the methods used in this study, and discussion questions are provided.

The HIV-1 matrix protein does not interact directly with the protein interactive domain of AP-3δ.

During the late phase of the Human Immunodeficiency Virus Type-1 (HIV-1) replication cycle, viral Gag proteins and the intact RNA genome are trafficked to specific sub-cellular membranes where virus assembly and budding occurs. Targeting to the plasma membranes of T cells and macrophages is mediated by interactions between the N-terminal matrix (MA) domain of Gag and cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] molecules. However, in macrophages and dendritic cells, a subset of Gag proteins appears to be targeted to tetraspanin enriched viral compartments, a process that appears to be mediated by MA interactions with the Delta subunit of the cellular Adaptor Protein AP-3 (AP-3δ). We cloned, overexpressed and purified the protein interactive domain of AP-3δ and probed for MA binding by NMR. Unexpectedly, no evidence of binding was observed in these in vitro experiments, even at relatively high protein concentrations (200µM), suggesting that AP-3δ plays an alternative role in HIV-1 assembly.