ALBERT

Description: Viral infection triggers a series of signaling cascades, which converge to activate the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3), thereby inducing the transcription of type I interferons (IFNs). Although not fully characterized, these innate antiviral responses are fine-tuned by dynamic ubiquitination and deubiquitination processes. In this study, we report ubiquitin-specific protease (USP) 15 is involved in regulation of the retinoic acid-inducible gene I (RIG-I)-dependent type I IFN induction pathway. Knockdown of endogenous USP15 augmented cellular antiviral responses. Overexpression of USP15 inhibited the transcription of IFN-β. Further analyses identified histidine 862 as a critical residue for USP15’s catalytic activity. Interestingly, USP15 specifically removed lysine 63-linked polyubiquitin chains from RIG-I among the essential components in RIG-I-like receptor-dependent pathway. In addition, we demonstrated that in contrast to USP15 de-ubiquitinating (DUB) activity, USP15-mediated inhibition of IFN signaling was not abolished by mutations eliminating the catalytic activity, indicating that a fraction of USP15-mediated IFN antagonism was independent of the DUB activity. Catalytically inactive USP15 mutants, as did the wild-type protein, disrupted virus-induced interaction of RIG-I and IFN-β promoter stimulator 1. Taken together, our data demonstrate that USP15 acts as a negative regulator of RIG-I signaling via DUB-dependent and independent mechanisms. Scientific Reports 5 doi: 10.1038/srep11220

Description: Most patients with idiopathic thrombotic thrombocytopenic purpura (TTP) are caused by acquired autoantibodies that bind and inhibit proteolytic activity of ADAMTS13, a metalloprotease that cleaves von Willebrand factor (vWF). However, the binding epitopes of these anti-ADAMTS13 autoantibodies in many cases have not been fully defined. The limited data in the literature were largely from studies of small sample size, using less desirable recombinant ADAMTS13 fragments expressed in E. coli bacteria or insect cells or employing a non-physiological technique for detection. The results were therefore either inconclusive or controversial. To overcome these pitfalls, the full-length ADAMTS13 and various fragments of ADAMTS13 were expressed in human embryonic kidney (HEK293) cells. The recombinant proteins were purified by a combination of anion exchange chromatography and Ni-NTA affinity column. The antibody and antigen reactions were all performed in phosphate-buffered saline at pH 7.4. The immune complexes were collected by incubation with protein A/G Sepharose. After electrophoresis under denatured and reduced conditions, the antigens (ADAMTS13 and fragments) were then detected by Western blot with anti-V5 IgG, which recognizes the carboxyl terminal V5- His epitopes of the recombinant proteins. We showed that IgGs from 67 patients with acquired idiopathic TTP all interacted with full-length ADAMTS13 (construct FL-AD13) and the CUB domainless variant (construct delCUB). Approximately 97% (65/67) of IgGs in TTP patients bound the variant truncated after the spacer domain (construct MDTCS). However, a removal of the Cys-rich and spacer domains (construct MDT) reduced its reactivity with IgGs from TTP patients by 88%, suggesting the important contribution of these domains to the antibody binding epitopes. Unexpectedly, a fragment consisting of a disintegrin domain, the first TSP1 repeat, the Cys-rich and spacer domains (construct DTCS) reacted with IgGs from only 52% of TTP patients, indicating the additional contribution of the metalloprotease domain to these non-linear binding epitopes. Moreover, approximately 31%, 37%, and 46% of patients’ IgGs targeted against the carboxyl terminal CUB domains, TSP1 2–8, and TSP1 5-8CUB, respectively. Low platelet count was observed at the time of admission in patients with the IgGs that reacted with the TSP1 2–8 and CUB domains of ADAMTS13, suggesting that the autoantibodies with the specificity toward more distal carboxyl-terminal domains of ADAMTS13 may impair ADAMTS13 function in vivo. We conclude that while the Cys-rich and spacer domains of ADAMTS13 contain the core epitopes, other adjacent functional domains may also contribute to the non-linear binding epitopes that are recognized by anti-ADAMTS13 IgGs. The data further support the role of autoantibodies against the middle and distal carboxyl terminal domains of ADAMTS13 in pathogenesis of TTP. Our findings provide novel insight into the mechanism of autoimmune-mediated TTP.

Description: Human prothrombinase assembled on synthetic membranes composed of phosphatidylcholine and phosphatidylserine (PCPS) catalyzes thrombin formation almost exclusively by sequential cleavage of prothrombin at Arg320 to yield the protease meizothrombin (mIIa) as an intermediate which is then further cleaved at Arg271. This cleavage pathway arises because Arg320 in intact prothrombin is cleaved ∼30-fold faster than Arg271. When prothrombin lacks γ-carboxyglutamic acid modifications (desGlaII), product formation is modestly decreased but bond cleavage largely occurs in the opposite order, yielding the zymogen, prethrombin 2 (P2), as an intermediate. This results from a reduction in the rate of cleavage at Arg320 in intact prothrombin and a partly compensating gain of function in cleavage at Arg271. Thus, membrane binding and/or other interactions mediated by γ-carboxyglutamic acids in the substrate play a major role in modulating the pathway for cleavage and whether the intermediate is a zymogen or protease. We now extend these approaches to evaluate prothrombinase function on activated platelets and human umbilical vein endothelial cells (HUVECs) at the physiologic concentration of prothrombin. Prothrombin activation was detected by measuring the appearance of product(s) with proteolytic activity and by analysis of the cleavage process by SDS-PAGE and western blot analysis quantitatively imaged by infrared fluorescence. This approach was validated by documenting equivalence in the progress curves obtained by western blot imaging or following staining of total protein in a purified system with PCPS membranes. Prothrombin cleavage was assessed using human platelets (108/ml), activated with thrombin and studied with saturating concentrations of Va and a limiting concentration of Xa. The pattern of prothrombin cleavage, intermediate accumulation and product formation was clearly distinct from that observed with PCPS membranes, indicating substantial flux towards thrombin formation via initial cleavage at Arg271 followed by cleavage at Arg320 producing the zymogen P2 as an intermediate. Transient formation of mIIa from initial cleavage at Arg320 was undetectable. Thus, prothrombinase assembled on thrombin activated platelets cleaves prothrombin in a way that is reminiscent of the cleavage of desGlaII rather than fully carboxylated prothrombin seen with PCPS membranes. In contrast, equivalent studies with prothrombinase assembled on thrombin activated HUVECs produced cleavage patterns consistent with significant flux towards thrombin formation via initial cleavage of prothrombin at Arg320 yielding mIIa as an intermediate. The use of desGlaII with HUVECs yielded lower rates and cleavage patterns similar to those obtained with fully carboxylated prothrombin in the platelet reactions. Our results document that the cleavage pathway for thrombin formation is dependent on cell type. Because mIIa is a protease with a different spectrum of activities from thrombin, its formation restricted to the vessel wall suggests an important regulatory role for the modulation of the pathway of prothrombin cleavage. Such bimodal regulation of the pathway for prothrombin cleavage on HUVECs and platelets suggests differential roles of prothrombinase assembled on platelets versus endothelium in regulating the hemostatic response to vascular injury.

Description: ADAMTS13 biosynthesis appeared to occur mainly in hepatic stellate cells, but detection of ADAMTS13 mRNA in many other tissues suggests that vascular endothelium may also produce ADAMTS13. We showed that ADAMTS13 mRNA and protein were detectable in human umbilical vein endothelial cells, aortic endothelial cells, and endothelium-derived cell line (ECV304). ADAMTS13 in cell lysate or serum-free conditioned medium cleaved von Willebrand factor (VWF) specifically. ADAMTS13 and VWF were localized to the distinct compartments of endothelial cells. Moreover, ADAMTS13 was preferentially sorted into apical domain of ECV304 and Madin-Darby canine kidney (MDCK) cells. Apical sorting of ADAMTS13 depended on the CUB domains and their association with lipid rafts. A mutation in the second CUB domain of ADAMTS13 (4143-4144insA), naturally occurring in patients with inherited thrombotic thrombocytopenic purpura, resulted in a significant reduction of ADAMTS13 secretion and a reversal of its polarity in MDCK cells. These data demonstrated that ADAMTS13 is synthesized and secreted from endothelial cells; the apically secreted ADAMTS13 from endothelial cells may contribute significantly to plasma ADAMTS13 proteases. The data also suggest a critical role of the CUB domains and a novel cargo-selective mechanism for apical sorting of a soluble ADAMTS protease in polarized cells.

Description: ADAMTS13, a metalloprotease that cleaves von Willebrand factor (vWF), is primarily synthesized in liver, endothelial cells and megakaryocytes/platelets. It circulates in plasma as an active protease with concentrations between 0.5 and 1.0 μg per milliliter of plasma. Proteolytic cleavage of endothelial cell bound and plasma vWF by ADAMTS13 is critical for maintaining normal hemostasis. Inability to cleave newly released unusually large vWF as a result of hereditary or acquired deficiency of ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Although plasma infusion or exchange is a proven effective therapy for TTP, the life-long plasma infusion for hereditary TTP is not without complications. In search for a better therapy, we performed an autologous transplantation of hematopoietic progenitor cells (HPCs) for correcting Adamts13 deficiency in a mouse model. The bone marrows were harvested from Adamts13−/− mice (C57BL6/129Sv) at 6–8 weeks of age and HPCs (CD48−/CD150+) were purified to a greater than 90% of purity in all cases using serial immune affinity depletion and enrichment techniques. The purified HPCs were transduced in culture with a self-inactivated lentiviral vector either encoding GFP or GFP plus murine full-length Adamts13 at 100 × MOI (multiplicity of infection) for 14–16 hours. The transduced HPCs (1 × 105) mixed with freshly prepared non-transduced bone marrow mononucleated cells (2 × 105) were then infused via tail vein into Adamts13−/− mice (recipients ) after lethal irradiation (450 cGy, twice, 3 h apart, at a dose rate of 2.2 cGy per minute). Peripheral blood was collected at 1, 3, and 5 months of post-transplantation for assessing the bone marrow chimerism and Adamts13 activity. The GFP positive cells were determined by flow cytometry and Adamts13 activity was determined by FRETS-vWF73 and GST-vWF73 peptides as described previously. We showed that all ten mice transplanted with the transduced HPCs encoding GFP plus murine Adamts13 exhibited persistent levels of plasma Adamts13 proteolytic activity, ranging from 10% to 60% of pooled normal murine plasma (PNP), despite low levels of bone marrow chimerism based on the percentage of GFP-positive mononucleated cells (~10–40%) in the peripheral blood. The expressed Adamts13 in plasma was able to cleave process unusually large vWF, resulting in a reduction of vWF multimers sizes compared with those in Adamts13−/− mice transplanted with HPCs transduced with the vector encoding GFP. Moreover, the levels of plasma Adamts13 appeared to be sufficient to protect mice against ferric chloride-induced carotid arterial thrombosis. The carotid arterial occlusion times for Adamts13−/− mice which did not undergo bone marrow transplantation were 5.6 ± 1.6 min, similar to those transplanted with HPCs transduced with the vector encoding GFP (4.3 ± 0.8 min) (mean ±SD) (p〉0.05). However, the carotid arterial occlusion times were significantly prolonged in Adamts13−/− mice transplanted with the vector encoding GFP plus murine Adamts13 (15.5 ± 6.8 min). The differences between the experimental group and the control groups were all statistically very significant (p

Description: ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13−/− mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride–induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.

Description: Degradation of newly released unusually large (UL)-vWF on endothelial cells by plasma ADAMTS13 metalloprotease is considered to be critical for maintaining normal hemostasis. An inability to degrade the cell bound string-like UL-vWF multimers by ADAMTS13 may result in excessive platelet adhesion and aggregation on cell surface, leading to thrombotic thrombocytopenic purpura (TTP). However, the requirement for shear stress and potential cofactors in this process is not fully defined. By immunofluorescent microscopy, we showed that upon histamine stimulation the newly released UL-vWF rapidly formed strings and bundles on human umbilical vein endothelial cells (HUVECs). These strings and bundles were removed within 2~5 minutes by plasma-derived and recombinant ADAMTS13 at concentrations of between 2.5 and 10 nM in 20 mM HEPES buffer, pH 7.5 containing 150 mM NaCl and 1 mM CaCl. This process did not appear to depend upon flow shear stress and coagulation factor VIII that has been shown to markedly increase the proteolytic cleavage of soluble vWF under high shear stress. The removal of the cell-bound UL-vWF strings and bundles correlated with the increases of vWF antigen and proteolytic cleavage fragments (176K and 140K) in the conditioned medium as determined by an enzyme-linked immunoassay and Western blot, respectively. The proteolytic cleavage of the cell bound UL-vWF by ADAMTS13 was time- and concentration-dependent, with a half maximal concentration of ADAMTS13 of approximately 5 nM. No cleavage product was detected when the histamine-stimulated HUVECs were incubated with the buffer alone or plasma from patients with acquired TTP and inhibitors or with heat-inactivated recombinant ADAMTS13. These results suggest that the degradation of cell bound UL-vWF is specific for ADAMTS13. Unexpectedly, the vWF multimers in the conditioned medium of ADAMTS13-treated endothelial cells were quite similar in sizes to those in the endothelial cells treated with histamine in the absence of ADAMTS13, indicating that the proteolytic cleavage of UL-vWF by ADASMTS13 occurs at the site proximal to cell membrane. Scanning electron microscopy clearly demonstrated that the breaking point of UL-vWF strings and bundles by ADAMTS13 was located at approximately 2~5 μm from the cell membrane. Structure-function analysis further demonstrated that the Cys-rich and spacer domains of ADAMTS13 were required for proteolytic cleavage of the cell-bound UL-vWF. The more distal carboxyl-terminal domains including TSP1 2–8 and CUB domains of ADAMTS13 appeared to be dispensable. We conclude that the cell bound UL-vWF polymers adopt an open conformation that is sensitive to ADAMTS13 proteolysis, analogous to those observed under the denaturing conditions. We hypothesize that membrane binding and/or interaction of UL-vWF bundles with certain scaffold proteins may provide an access of ADAMTS13 metalloprotease to the cleavage (Tyr1605-Met1606) bond at the central A2 domain of the vWF proximal to cell surface. These findings provide novel insight into the biological function of ADAMTS13 in processing UL-vWF and help understand the pathogenesis of TTP.

Description: ADAMTS13, a reprolysin-like metalloprotease, limits platelet aggregation by cleaving von Willebrand factor. Deficiency of plasma ADAMTS13 activity can lead to a life-threatening complication, thrombotic thrombocytopenic purpura (TTP). It has been shown that ADAMTS13 is synthesized in human and mouse hepatic stellate cells. But detection of ADAMTS13 mRNA by a reverse transcriptase polymerase chain reaction (RT-PCR) in many other tissues (brain, heart, lungs, kidneys, pancreas, adrenal gland, prostate, uterus and placenta) suggests that the vascular endothelial cells may also produce low level of ADAMTS13 protease. We showed that full-length ADAMTS13 mRNA and protein were detected in human umbilical cord vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), and microvascular endothelial cells (ECV304) by RT-PCR, SDS-polyacrylamide gel electrophoresis and Western blot. The ADAMTS13 in the cell lysates and the serum-free conditioned media of HUVEC, HAEC and ECV304 cleaved a peptidyl substrate, FRETS-VWF73, specifically, and such a proteolytic cleavage could be completely abolished by 10 mM EDTA. Immunohistochemistry showed that both endogenous and recombinant ADAMTS13 in HUVEC and HAEC appeared to be co-localized with von Willebrand factor in the endoplasmic reticulum and/or Golgi apparatus, but not in the Weibel-Parade bodies, suggesting that ADAMTS13 is readily secreted rather than stored inside the cells. To determine whether ADAMTS13 is secreted into the lumen of the vessels, we assessed the polarity of ADAMTS13 secretion in ECV304 and Madin-Darby canine kidney (MDCK) cells, two well-characterized polarized endothelial and epithelial cell lines for studying protein sorting. Both endogenous ADAMTS13 and recombinant ADAMTS13-V5-His expressed in ECV304 was predominantly secreted into the apical domain of the cells. Similarly, the recombinant ADAMTS13-V5-His in MDCK cells was also secreted predominantly into the apical domain. Apical secretion of ADAMTS13-V5-His appeared to depend on at least a transient association of ADAMTS13-V5-His with Triton X-100 insoluble glycosphingolipid and cholesterol-enriched microdomains, namely rafts. Deletion of the distal carboxyl terminal CUB (Complement C1r/C1s, Urinary EGF, and Bone morphogenic protein) domains or removal of membrane cholesterol by treatment of MDCK cells expressing recombinant ADAMTS13-V5-His with lovastatin and methyl-β-cyclodextrin completely abolished the raft association and apical secretion of ADAMTS13-V5-His in MDCK cells. Moreover, an ADAMTS13 mutation (4143insA), which naturally occurs in a patient with TTP that deletes the second CUB domain not only significantly impaired the amount of total ADAMTS13 protein secreted into the conditioned medium, but also reversed the polarity of ADAMTS13 secretion in MDCK cells. Our data demonstrate that: 1) active full-length ADAMTS13 protease is produced in human vascular endothelial cells; 2) the CUB domains are critical for apical secretion via their interaction with lipid rafts; 3) mutations in ADAMTS13 gene that disrupt sorting signals may result in congenital ADAMTS13 deficiency, leading to TTP in some cases. Considering a large surface area of the vascular endothelial cells, apically secreted ADAMTS13 from these cells may contribute significantly to plasma pool of ADAMTS13 protease.