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.
Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ...ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen–deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.
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.
•Describe human MK populations representing distinct developmental stages within a heterogeneous culture.•FV uptake identifies cultured MKs ready to release platelets upon infusion into mice.
Platelet dense granules (DGs) are storage organelles for calcium ions, small organic molecules such as adenosine 5′-diphosphate and serotonin, and larger polyphosphates that are secreted upon ...platelet stimulation to enhance platelet activation, adhesion, and stabilization at sites of vascular damage. DGs are thought to fully mature within megakaryocytes (MKs) prior to platelet formation. Here we challenge this notion by exploiting vital fluorescent dyes to distinguish mildly acidic DGs from highly acidic compartments by microscopy in platelets and MKs. In isolated primary mouse platelets, compartments labeled by mepacrine, a fluorescent weak base that accumulates in DGs, are readily distinguishable from highly acidic compartments, likely lysosomes, that are labeled by the acidic pH indicator LysoTracker and from endolysosomes and α granules labeled by internalized and partially digested self-quenching BODIPY–labeled bovine serum albumin (DQ BSA). By contrast, in murine fetal liver and human CD34+ cell-derived MKs and the megakaryocytoid cell lines, MEG-01 and differentiated G1ME2, labeling by mepacrine overlapped nearly completely with labeling by LysoTracker and partially with labeling by DQ BSA. Mepacrine labeling in G1ME2-derived MKs was fully sensitive to proton ATPase inhibitors but was only partially sensitive in platelets. These data indicate that mepacrine in MKs accumulates as a weak base in endolysosomes but is likely pumped into or retained in separate DGs in platelets. Fluorescent puncta that labeled uniquely for mepacrine were first evident in G1ME2-derived proplatelets, suggesting that DGs undergo a maturation step that initiates in the final stages of MK differentiation.
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•Compartments labeled by the vital dense granule dye mepacrine are distinct from acidic structures in platelets but not in megakaryocytes.•Distinct hypoacidic mepacrine-labeled structures first appear during megakaryocyte differentiation to proplatelets.
The regulation of von Willebrand factor (VWF) processing, packaging, and secretion is important for primary hemostasis. Recently, autophagy has been implicated in modulating VWF maturation and ...Weibel-Palade body (WPB) morphology. Additionally, treatment of mice and humans with chloroquine (CQ), an anti-malarial agent and pharmacological inhibitor of autophagy, is shown to increase bleeding time. Therefore, we hypothesize that targeting autophagic flux may be therapeutic for arterial thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP). To test this hypothesis, we first treated human umbilical vein endothelial cells (HUVECs) with CQ in culture and showed that CQ dose-dependently decreased VWF antigen levels and multimer sizes in the conditioned medium of histamine-stimulated cells (not shown). Knockdown of an autophagy-related protein (Atg7) with shRNA in HUVECs had similar effect to CQ treatment in VWF secretion and multimer distribution (not shown). More interestingly, daily injections (i.p.) of CQ at 60 mg/kg into Adamts13-/- mice (CAST/Ei strain) for 7 days reduced plasma VWF concentration by more than 60% compared to vehicle control (Fig. 1). Interestingly, VWF secreted from CQ-treated mice remained functional as collagen binding activity/antigen ratio was comparable to vehicle control mice. However, multimer analysis demonstrated the selective lack of ultra large VWF in plasma of Adamts13-/- mice treated with CQ as compared with those treated with vehicle alone (Fig. 2). These results indicate that targeting autophagy pathway with a pharmacological agent, such as CQ, may modulate VWF secretion and, thereby, arterial thrombosis. Our ongoing experiments are to determine the therapeutic efficacy of CQ and other autophagy inhibitors in murine models of arterial thrombosis and TTP.
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No relevant conflicts of interest to declare.
Hermansky‐Pudlak syndrome (HPS) is a group of rare genetic diseases characterized by oculocutaneous albinism and bleeding diathesis, often associated with lung fibrosis. The symptoms of HPS reflect ...defects in membrane transport processes required to generate tissue‐specific lysosome‐related organelles (LROs), such as melanosomes in pigment cells, lamellar bodies (LB) in type 2 alveolar epithelial cells (AT2) and dense granules in platelets. Although the transport processes that are defective in HPS are shared among distinct LRO‐expressing cells, it is not clear whether the unique cargo proteins that confer distinct morphological and functional characteristics are targeted to their respective LROs by conserved or cell type‐specific recognition mechanisms. To determine whether LB and melanosome cargo proteins contain cell type‐independent LRO sorting information, we exploited the behavior of model cargo proteins, such as melanosomal protein, OCA2 and LB‐specific protein, ATP8A1 in AT2 cells and melanocytes. OCA2 is a pigment cell‐specific membrane protein and localizes to melanosomes via a cytoplasmic dileucine‐based sorting signal that engages both AP‐1 and AP‐3. By overexpression of wild‐type OCA2 in MLE15, we show that wild‐type OCA2 colocalized extensively with the LB marker, mCherry‐ABCA3 in MLE15, an AT2 cell line. OCA2‐AA23N bearing the double E96DP99S mutation that was unable to bind to AP‐1 and AP‐3 localized to the cell surface. To confirm the relative roles of AP‐1 and AP‐3 in transport of OCA2 to LB, we also expressed the AP‐3‐biased OCA2‐AA23‐P99S and AP‐1‐biased OCA2‐AA23‐E96D variants in MLE15. Mutants that preferentially bound either AP‐1 or AP‐3 each partially trafficked toward LB, but AP‐3 binding was necessary for steady‐state LB localization, as it is for delivery of OCA2 to melanosomes. ATP8A1, a member of the P4 subfamily of P‐type ATPases (P4‐ATPases), is highly enriched in LB but expressed at low levels in melanocytes. When expressed in immortalized mouse melan‐Ink4a−/− melanocytes, an EGFP‐tagged, catalytically inactive ATP8A1 partially overlapped with pigmented melanosomes. Overlap was substantially reduced by mutagenesis of a conserved dileucine‐based sorting signal or by expression in AP‐3‐deficient melanocytes. These data provide evidence that cellular machinery in AT2 or melanocytes can recognize a universal LRO targeting signal in a heterologous protein and deliver it to a cell type‐specific LRO. By contrast, two other AT2‐restricted LB cargoes – DC‐LAMP and ABCA3 ‐ failed to localize to pigmented melanosomes upon expression in melan‐Ink4a−/− cells. Together, these data suggest that LRO cargoes employ both universally conserved and cell type‐specific pathways for delivery to nascent LROs.
Support or Funding Information
NIH (GM108807/SG and MM). SG is the Julia Carell Stadler Professor of Pediatrics.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Alveolar type 2 cells (AT2) are involved in the maintenance of epithelial homeostasis and repair after injury. Lamellar bodies (LB) in AT2 play a critical role in lung health and disease as the site ...of surfactant synthesis, packaging and secretion. LB biogenesis is poorly understood, especially mechanisms that foster membrane and protein trafficking to LB. In this study, we found that ATP8a1, a member of the P4 subfamily of P‐;type ATPases (P4‐;ATPases), was highly enriched in the ABCA3‐positive LB fraction derived from human fetal lung explants. In AT2 from mice lacking the adaptor protein 3 complex (AP‐3), the pearl mouse model of Hermansky Pudlak syndrome type 2 (HPS2), ATP8a1 was largely depleted from ABCA3‐positive LB and accumulated in Rab11‐positive recycling endosomes (REs). Using targeted mutagenesis, we identified a di‐leucine‐based sorting signal (ExxxLL) present in the C‐terminal cytoplasmic domain of ATP8a1 that is necessary for its proper targeting to LB of AT2. To dissect the relative roles of AP‐1 and AP‐3 in the transport of ATP8a1 to LB, we have developed MLE‐15 cell lines in which a subunit of AP‐1 (Ap1γ1) and/or AP‐3 (Ap3β1) has been inactivated using CRISPR/Cas9 gene editing and tested the targeting of ATP8a1 in these mutated cell lines. The lack of AP‐3 in MLE‐15 reduced efficient LB delivery of ATP8a1, while deletion of the AP‐1 subunit had no effect on ATP8a1 delivery to LB. P4‐;ATPases typically flip aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of organelle membranes. PS is highly enriched in REs and is essential for endosomal membrane traffic. Here, we show that the cytoplasmic leaflet of LB in MLE‐15 and human AT2 are highly enriched in PS using a GFP‐LactC2 probe; in contrast, AP‐3‐deficient cells showed impaired distribution of PS, favoring RE locazliation. These data provide evidence that ATP8a1 is dependent on AP‐3 for trafficking from RE to LB in AT2, and suggest that loss of ATP8a1 targeting to LB in HPS2 impairs PS localization to LB in AT2 cells.
Support or Funding Information
National Institutes of Health (GM108807/SG, MM and LG). SG is the Julia Carell Stadler Professor of Pediatrics.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Here, we provide a comprehensive review of current findings concerning the biochemistry and physiological functions of ADAMTS7, a metalloprotease that is known to interact with cartilage oligomeric ...matrix protein, progranulin, and alpha2-macroglobulin. Such broad substrate specificity and potentially diverse physiological functions make ADAMTS7 an interesting enzyme to study. ADAMTS7 has been shown to play a role in the pathogenesis of arthritis and disc disorders. More recently, the
locus is identified to have a strong association with coronary atherosclerotic disease. However, the role of ADAMTS7 in the development of atherosclerosis is yet to be determined. The development of an easy and high throughput assay for ADAMTS7 activity and appropriate animal models will allow us to uncover the novel mechanisms of coronary arterial disease.
ADAM28, a member of ADAM (A Disintegrin and Metalloprotease) family, consists of two isoforms: a membrane-type form and a secreted form. Both ADAM28 isoforms are expressed in normal T and B ...lymphocytes of peripheral blood and lymphatic organs and certain malignant tumor cells. ADAM28 exhibits catalytic activity toward a few substrates including insulin-like growth factor binding protein-3 (IGFBP-3) and von Willebrand factor (VWF). ADAM28 is also overexpressed in human non-small cell lung carcinomas and breast carcinomas, predominantly by maligant cells, and the expression levels correlate with malignant cell proliferation and lymph node metastasis, presumably mediated by the cleavage of VWF. However, the structural components of ADAM28 required for proteolytic cleavage of VWF are not known. In this study, we constructed and expressed a soluble murine ADAM28 lacking the transmembrane domain and cytoplasmic tail (ΔTMA28) and various other carboxyl-terminal truncated ADAM28 variants containing a metalloprotease domain (M), plus a disintegrin domain (MD), and a Cys-rich domain (MDC) in COS-7 cells. All constructs were tagged at their carboxyl termini with a 3xflag epitope. Conditioned medium from transfected cells was collected and concentrated by a filtration technique. Protein concentrations of each construct in the concentrated conditioned medium were determined by a semi-quantitative Western blotting using a monoclonal anti-Flag IgG. A carboxyl-terminal truncated ADAMTS13 fragment (MDTCS) containing a 3xFlag epitope was used for a calibration. The proteolytic cleavage of a murine recombinant VWF (mVWF) by murine recombinant ADAM28 variants was carried out at 37 °C in a buffer containing 1 µM ZnCl2 and 5 mM CaCl2 in the presence or in the absence of guanidine-HCl at neutral pH 7.5. A full-length murine recombinant ADAMTS13 (mFL-A13) was used as a positive control and the conditioned medium derived from untransfected cells was used for a negative control. We show that mVWF that is pre-denatured with 1.0 M guanidine-HCl (at 37 °C for 2 hours) was efficiently cleaved by the constructs M, MD, MDC, and ΔTMA28 at the concentration of 50-100 nM after 18 hours of incubation. The proteolytic cleavage of VWF by ADAMTS28 variants results in dramatically reduced immune reactivity with rabbit anti-VWF IgG and multimer size as demonstrated by SDS-agarose gel (1%) electrophoresis and Western blotting. Paradoxically, this cleavage significantly increases the VWF-collagen binding activity despite of smaller VWF multimer size, suggesting that ADAM28-mediated proteolysis of VWF exposes its high-affinity collagen binding site. The cleavage efficiency of mVWF by all ADAM28 variants is comparable to that by mFL-A13 under the same conditions. The cleavage of native mVWF by ADAM28 is much less than that of pre-denatured VWF. EDTA (10 mM) added to the reaction completely abolishes the proteolytic cleavage of mVWF by all mADAM28 variants. We conclude that a metalloprotease domain of mADAM28 is sufficient for proteolytic cleavage of mVWF. The carboxyl terminal ancillary domains of mADAM28 appear to be dispensable for recognition and cleavage of mVWF. Our findings suggest a different mechanism underlying the proteolysis of VWF by a ADAM family protease. Further investigation into the biological activity of ADAM28 variants under more physiological conditions may shed new light on the mechanism of ADAM and ADAMTS proteolysis. The findings may provide a potential novel therapy for arterial thrombosis and acquired thrombotic thrombocytopenic purpura due to autoantibody inhibition because ADAM28 activity is not expected to be inhibited by ADAMTS13-specific autoantibodies.
No relevant conflicts of interest to declare.
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