A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken.

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.

AP-3-dependent targeting of flippase ATP8A1 to lamellar bodies suppresses activation of YAP in alveolar epithelial type 2 cells.

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.

PTEN functions by recruitment to cytoplasmic vesicles.

PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.

Phosphatidylinositol-4-kinase IIα licenses phagosomes for TLR4 signaling and MHC-II presentation in dendritic cells.

Toll-like receptor (TLR) recruitment to phagosomes in dendritic cells (DCs) and downstream TLR signaling are essential to initiate antimicrobial immune responses. However, the mechanisms underlying TLR localization to phagosomes are poorly characterized. We show herein that phosphatidylinositol-4-kinase IIα (PI4KIIα) plays a key role in initiating phagosomal TLR4 responses in murine DCs by generating a phosphatidylinositol-4-phosphate (PtdIns4P) platform conducive to the binding of the TLR sorting adaptor Toll-IL1 receptor (TIR) domain-containing adaptor protein (TIRAP). PI4KIIα is recruited to maturing lipopolysaccharide (LPS)-containing phagosomes in an adaptor protein-3 (AP-3)-dependent manner, and both PI4KIIα and PtdIns4P are detected on phagosomal membrane tubules. Knockdown of PI4KIIα-but not the related PI4KIIß-impairs TIRAP and TLR4 localization to phagosomes, reduces proinflammatory cytokine secretion, abolishes phagosomal tubule formation, and impairs major histocompatibility complex II (MHC-II) presentation. Phagosomal TLR responses in PI4KIIα-deficient DCs are restored by reexpression of wild-type PI4KIIα, but not of variants lacking kinase activity or AP-3 binding. Our data indicate that PI4KIIα is an essential regulator of phagosomal TLR signaling in DCs by ensuring optimal TIRAP recruitment to phagosomes.

The road to lysosome-related organelles: Insights from Hermansky-Pudlak syndrome and other rare diseases.

Lysosome-related organelles (LROs) comprise a diverse group of cell type-specific, membrane-bound subcellular organelles that derive at least in part from the endolysosomal system but that have unique contents, morphologies and functions to support specific physiological roles. They include: melanosomes that provide pigment to our eyes and skin; alpha and dense granules in platelets, and lytic granules in cytotoxic T cells and natural killer cells, which release effectors to regulate hemostasis and immunity; and distinct classes of lamellar bodies in lung epithelial cells and keratinocytes that support lung plasticity and skin lubrication. The formation, maturation and/or secretion of subsets of LROs are dysfunctional or entirely absent in a number of hereditary syndromic disorders, including in particular the Hermansky-Pudlak syndromes. This review provides a comprehensive overview of LROs in humans and model organisms and presents our current understanding of how the products of genes that are defective in heritable diseases impact their formation, motility and ultimate secretion.

Adaptor protein-3 in dendritic cells facilitates phagosomal toll-like receptor signaling and antigen presentation to CD4(+) T cells.

Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic Toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4(+) T cell activation and Th1 effector cell function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores.

BLOC1S5 pathogenic variants cause a new type of Hermansky-Pudlak syndrome.

PURPOSE: Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, excessive bleeding, and often additional symptoms. Variants in ten different genes have been involved in HPS. However, some patients lack variants in these genes. We aimed to identify new genes involved in nonsyndromic or syndromic forms of albinism. METHODS: Two hundred thirty albinism patients lacking a molecular diagnosis of albinism were screened for pathogenic variants in candidate genes with known links to pigmentation or HPS pathophysiology. RESULTS: We identified two unrelated patients with distinct homozygous variants of the BLOC1S5 gene. Patients had mild oculocutaneous albinism, moderate bleeding diathesis, platelet aggregation deficit, and a dramatically decreased number of platelet dense granules, all signs compatible with HPS. Functional tests performed on platelets of one patient displayed an absence of the obligate multisubunit complex BLOC-1, showing that the variant disrupts BLOC1S5 function and impairs BLOC-1 assembly. Expression of the patient-derived BLOC1S5 deletion in nonpigmented murine Bloc1s5-/- melan-mu melanocytes failed to rescue pigmentation, the assembly of a functional BLOC-1 complex, and melanosome cargo trafficking, unlike the wild-type allele. CONCLUSION: Mutation of BLOC1S5 is disease-causing, and we propose that BLOC1S5 is the gene for a new form of Hermansky-Pudlak syndrome, HPS-11.

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-ß1 secretion and hematopoiesis in mice.

We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα-/- and pitpα-/-/ß-/-) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα-/-/ß-/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα-/- and pitpα-/-/ß-/- BM MKs contained higher levels of transforming growth factor ß1 (TGF-ß1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα-/- and pitpα-/-/ß-/- mice had higher concentrations of TGF-ß1. CM from pitpα-/- and pitpα-/-/ß-/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-ß antibody, and treatment of pitpα-/-/ß-/- mice in vivo with anti-TGF-ß antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-ß and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα-/-/ß-/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-ß, are stored in MK α-granules and were also elevated in CM of cultured pitpα-/-/ß-/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-ß1.

Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity.

Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 -which optimizes toll-like receptor signaling from phagosomes-sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to Salmonella Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1ß and IL-18 in response to particulate stimuli in vitro, but caspase-1 and IL-1ß levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1ß, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients.

Identifying and enriching platelet-producing human stem cell-derived megakaryocytes using factor V uptake.

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

Melanosomes--dark organelles enlighten endosomal membrane transport.

Melanosomes are tissue-specific lysosome-related organelles of pigment cells in which melanins are synthesized and stored. Analyses of the trafficking and fate of melanosomal components are beginning to reveal how melanosomes are formed through novel pathways from early endosomal intermediates. These studies unveil generalized structural and functional modifications of the endosomal system in specialized cells, and provide unexpected insights into the biogenesis of multivesicular bodies and how compartmentalization regulates protein refolding. Moreover, genetic disorders that affect the biogenesis of melanosomes and other lysosome-related organelles have shed light onto the molecular machinery that controls specialized endosomal sorting events.

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation.

The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils.

Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

Defective release of α granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models.

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.

Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale.

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.

TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation.

Dendritic cells (DCs) phagocytose large particles like bacteria at sites of infection and progressively degrade them within maturing phagosomes. Phagosomes in DCs are also signaling platforms for pattern recognition receptors, such as Toll-like receptors (TLRs), and sites for assembly of cargo-derived peptides with major histocompatibility complex class II (MHC-II) molecules. Although TLR signaling from phagosomes stimulates presentation of phagocytosed antigens, the mechanisms underlying this enhancement and the cell surface delivery of MHC-II-peptide complexes from phagosomes are not known. We show that in DCs, maturing phagosomes extend numerous long tubules several hours after phagocytosis. Tubule formation requires an intact microtubule and actin cytoskeleton and MyD88-dependent phagosomal TLR signaling, but not phagolysosome formation or extensive proteolysis. In contrast to the tubules that emerge from endolysosomes after uptake of soluble ligands and TLR stimulation, the late-onset phagosomal tubules are not essential for delivery of phagosome-derived MHC-II-peptide complexes to the plasma membrane. Rather, tubulation promotes MHC-II presentation by enabling maximal cargo transfer among phagosomes that bear a TLR signature. Our data show that phagosomal tubules in DCs are functionally distinct from those that emerge from lysosomes and are unique adaptations of the phagocytic machinery that facilitate cargo exchange and antigen presentation among TLR-signaling phagosomes.

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells.

Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aß peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.

Presentation of phagocytosed antigens by MHC class I and II.

Phagocytosis provides innate immune cells with a mechanism to take up and destroy pathogenic bacteria, apoptotic cells and other large particles. In some cases, however, peptide antigens from these particles are preserved for presentation in association with major histocompatibility complex (MHC) class I or class II molecules in order to stimulate antigen-specific T cells. Processing and presentation of antigens from phagosomes presents a number of distinct challenges relative to antigens internalized by other means; while bacterial antigens were among the first discovered to be presented to T cells, analyses of the cellular mechanisms by which peptides from phagocytosed antigens assemble with MHC molecules and by which these complexes are then expressed at the plasma membrane have lagged behind those of conventional model soluble antigens. In this review, we cover recent advances in our understanding of these processes, including the unique cross-presentation of phagocytosed antigens by MHC class I molecules, and in their control by signaling modalities in phagocytic cells.