Acquired thrombotic thrombocytopenic purpura (TTP), a potentially fatal arterial thrombotic disorder, is primarily caused by autoantibodies that bind and inhibit plasma von Willebrand factor ...(VWF)-cleaving metalloprotease (ADAMTS13) activity. However, the mechanisms underlying autoantibody-mediated inhibition of ADAMTS13 activity and acquired TTP are not fully understood. By a hydrogen-deuterium exchange coupled with mass spectrometry (HX-MS) technique, we found that human monoclonal anti-ADAMTS13 antibodies, the single chain variable region fragments (scFvs)4-20, 4-16, and 3-1 that were, isolated by phage display from two patients with acquired TTP, predominantly bound to a discontinuous and conformational epitope in the spacer domain of ADAMTS13 with a subtle difference. The epitope for scFvs4-20 and 4-16 comprises five small flexible loops, including a previously described motif A (or exosite 3, R659-E664), motif B (exosite 4, L632-R639), and several other outlying residues (F592, Y658, and Y665), while scFv3-1 bound all other residues except for those in motif A. Site-directed mutagenesis and biochemical analysis demonstrated that both motifs A and B were found to be critical for recognition and proteolysis of VWF73 and multimeric VWF. Deletion of motif A or motif B in full-length ADAMTS13 abolished the binding of scFvs4-20 and 4-16 but not 3-1 (which did not bind motif A). Our findings demonstrate the powerful use of HX-MS for mapping antibody epitopes at nearly single amino acid resolution. This provides a new way to reveal mechanisms of autoantibody-mediated inhibition of plasma ADAMTS13 activity and acquired TTP.
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No relevant conflicts of interest to declare.
ADAM28, a member of the ADAM (A Disintegrin And Metalloproteinase) family, cleaves various substrates including von Willebrand factor. Two isoforms of ADAM28: the membrane associated (ADAM28m) and ...the secreted (ADAM28s) were identified. Both were overexpressed in solid tumors including breast carcinoma, non-small cell lung cancer, and bladder transitional cell carcinoma. Overexpression of ADAM28 has been shown to promote cell growth, invasion, and metastasis of solid tumors in humans. However, little is known about expression and potential role of ADAM28 in hematological malignancies. In this study, we determined ADAM28 protein and/or mRNA expression in plasma and/or bone marrow cells of patients with various acute and chronic leukemia. An informed consent was obtained from all participants. Plasma and bone marrow aspirates were obtained from a total of 65 patients including 24 patients with acute lymphoblastic leukemia (ALL), 23 patients acute myelocytic leukemia (AML), and 18 patients with other leukemia. Twenty one healthy volunteers were also included in the study (Table 1). By flow cytometry, we showed that mean fluorescent index ratios that are specific for ADAM28m were significantly <>
<>increased in the bone marrow malignant cells in patients with ALL (2.9±0.13) and AML (2.1±0.17) compared with those in the control (0.4±0.06). The p values were <0.01 and <0.05, respectively. In addition, plasma levels of ADAM28s antigen assessed by a specific sandwich ELISA were significantly higher in patients with ALL (386.1±44.6 pg/ml) (p<0.01) and AML (291.3±24.0 pg/ml) (p<0.05) than those in the control (120.7±12.1 pg/ml). Furthermore, the mRNA levels encoding ADAM28m (1.7±0.0) and ADAM28s (2.9±0.1) in the bone marrow cells were significantly higher than those in the control (p< 0.05) (Table 1). Interestingly, cellular ADAM28 overexpression was inversely associated with cellular apoptosis in ALL patients. In four ALL patients with an early relapse, ADAM28 levels (both protein and mRNA) in bone marrow cells were significantly higher than those in patients with complete remission. In conclusion, our data indicate for the first time that a measurement of plasma and bone marrow ADAM28 levels may help identify patients with high risk of early relapse. Whether a treatment that targets ADAM28 has an effect in adult acute leukemia remains to be determined. Display omitted
No relevant conflicts of interest to declare.
Acquired thrombotic thrombocytopenic purpura (aTTP), a potentially fatal syndrome, is primarily caused by autoantibodies against the metalloprotease ADAMTS13. Most patients with aTTP harbor an ...immunoglobulin (Ig) G isotype in blood that targets the spacer domain of ADAMTS13. The precise epitopes of the anti-ADAMTS13 IgGs and the mechanism underlying their inhibition activity are not fully understood. We hypothesized that inhibitory IgG autoantibodies from aTTP patients achieve their inhibitory function by binding to a discontinuous epitope in the spacer domain of ADAMTS13. To test this hypothesis, we determined the binding epitope of one out of >100 unique human monoclonal antibody (mAb) fragments (single-chain Fv, scFv) isolated by phage display from aTTP patients. We developed a novel hydrogen-deuterium exchange-mass spectrometry technology (HX-MS) to identify the antibody binding sites at single amino acid residue resolution. Human ADAMTS13 inhibitory scFv 4-20 was expressed in E. coli Top10 cells and purified to homogeneity by Ni-chelating affinity chromatography. In the HX-MS experiment, the mAb was coupled to affi-gel 10 resin and used to bind recombinant ADAMTS13-MDTCS fragment expressed in a stably transfected Drosophila schneider 2 (S2) cell line. After exchange with deuterium (D2O) oxide for various periods of time, the reaction was stopped, the protein was eluted, and digested to peptide fragments with pepsin, and the peptides with or without deuterium bound were resolved and identified by fast HPLC and mass spectrometry. We find that mAb scFv4-20 binds to amino acid residues Arg636, Leu637, Arg639, and Leu640 spanning from Leu632 to Leu640 (in exosite 4) in the spacer domain of ADAMTS13. This sequence is highly conserved in the ADAMTS13 spacer domains from zebrafish to mammals. In addition, mAb scFv4-20 binds Arg660, Tyr661, and Tyr665 in exosite 3, previously shown to play an important role in substrate recognition and anti-ADAMTS13 autoantibody-mediated inhibition, as well as Lys608, upstream exosites 3 and 4. Apparently, mAb scFv4-20 inhibits plasma ADAMTS13 activity (IC50 ∼0.40 nM) by binding these non-linear surface residues in the spacer domain (Fig. 1A). In agreement, site-directed mutagenesis shows that complete deletion (Δ632LTEDRLPR639) or partial deletion (Δ632LTED635 or Δ636RLPR639), or replacement of these residues with alanines (632LTED635/4A or 636RLPR639/4A) abolished or dramatically reduced mAb scFv4-20 binding. A deletion or alanine substitution of the surface residues on exosite 4 also abolished or reduced ADAMTS13 proteolytic activity toward a fluorescein-labeled VWF73 peptide and multimeric VWF (Fig. 1B), indicating that the ADAMTS13 epitope for mAb scFv4-20 is also part of ADAMTS13's substrate recognition site. We conclude that anti-ADAMTS13 autoantibodies work by physically blocking the well-conserved VWF binding site on ADAMTS13. These results demonstrate the powerful use of HX-MS technology to determine both linear and non-linear antibody binding epitopes. The results provide valuable information concerning the mechanism of autoantibody-mediated aTTP that may be exploited to develop targeted therapy by reengineering ADAMTS13 to avoid autoantibody inhibition. Display omitted
No relevant conflicts of interest to declare.
It is not clear how the pathology, presentation and outcome for patients who present with de novo metastatic breast cancer (dnMBC) compare with those who later develop distant metastases. DnMBC is ...uncommon in younger patients. We describe these differences within a cohort of young patients in the United Kingdom.
Women aged 40 years or younger with a first invasive breast cancer were recruited to the prospective POSH national cohort study. Baseline clinicopathological data were collected, with annual follow-up. Overall survival (OS) and post-distant relapse-free survival (PDRS) were assessed using Kaplan-Meier curves.
In total, 862 patients were diagnosed with metastatic disease. DnMBC prevalence was 2.6% (76/2977). Of those with initially localised disease, 27.1% (786/2901) subsequently developed a distant recurrence. Median follow-up was 11.00 years (95% CI 10.79-11.59). Patients who developed metastatic disease within 12 months had worse OS than dnMBC patients (HR 2.64; 1.84-3.77). For PDRS, dnMBC was better than all groups, including those who relapsed after 5 years. Of dnMBC patients, 1.3% had a gBRCA1, and 11.8% a gBRCA2 mutation.
Young women with dnMBC have better PDRS than those who develop relapsed metastatic breast cancer. A gBRCA2 mutation was overrepresented in dnMBC.
Platelet dense granules (DGs) are storage organelles for calcium ions, small organic molecules such as ADP and serotonin, and larger polyphosphates that are secreted upon platelet stimulation to ...enhance platelet activation, adhesion, and stabilization at sites of vascular damage. However, the precise timing of DG formation and maturation has not been definitively characterized, and how and when DG membrane contents are delivered is not at all known. In this thesis, I will discuss two important findings relevant to DG biology. The first finding addresses the timing of maturation of DGs within differentiation of the platelet precursor cell, the megakaryocyte (MK). DGs are thought to fully mature within MKs prior to platelet formation. Here I challenge this notion by exploiting vital fluorescent dyes to distinguish mildly acidic DGs from highly acidic compartments in platelets and MKs. In 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 alpha (α) granules. By contrast, in primary MKs and megakaryocytoid cell lines, MEG-01 and differentiated G1ME2, labeling by mepacrine overlapped nearly completely with labeling by LysoTracker. 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. The second finding addresses the putative DG transmembrane protein vesicular monoamine transporter 2 (VMAT2) and its localization throughout MK differentiation. VMAT2 has been implicated as the transporter responsible for serotonin uptake into DGs and is therefore a likely candidate for a DG protein. Within MKs, VMAT2 localizes to acidic endolysosomal compartments and partially to CD63-containing structures. VMAT2 does not localize to early endosomes. However, as G1ME2 MKs differentiate to proplatelets, VMAT2 cosegregates with mepacrine from acidic endolysosomes, likely multivesicular bodies, suggesting that DG membrane sorting occurs during proplatelet differentiation. VMAT2 does not bind to adaptor protein (AP) complexes, but may employ an acidic cluster to effect localization. Together, these two studies provide insight into the timing and maturation of DG biogenesis during MK differentiation.
Abstract 2214
Exosite binding plays a key role in cleavage of VWF by ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13). Two exosites that are evolutionarily ...conserved from zebra fish to mammals have been identified in the spacer domain by sequence alignment. Previous studies have shown that exosite 3 in the spacer domain plays a critical role for substrate recognition (Blood 115:2300–10, 2010), and modification of this exosite generates ADAMTS13 variants with improved specific activity but reduced autoantibody binding (Blood 119:3836–43, 2012). In the present study, using a site-directed mutagenesis approach, we identified a novel exosite near exosite 3 in the spacer domain, termed exosite 4, a region between residues Glu634 and Arg639. A partial (ΔEx4a:deletion of Leu632-Asp635 or ΔEx4b:deletion of Arg636-Arg639) or complete deletion of the exosite (ΔEx4) significantly impaired proteolytic activity towards peptidyl VWF73 and multimeric VWF. Moreover, substitution of all surface exposed residues in Ex4A (LTED/AAAA) or Ex4b (RLPR/AAAA) with alanine had a similarly detrimental effect on proteolytic activity. Further studies demonstrated that the residues Asp635 and Arg636 in exosite 4 play a critical role for substrate recognition. We conclude that the region between residues Glu634 and Arg639 is a novel exosite necessary for recognition and cleavage of VWF.
No relevant conflicts of interest to declare.
Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin ...modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.
•BET protein Brd2a is a bi-modal developmental regulator of apoptosis in zebrafish•Brd2a is required for proper formation of the vertebrate nervous system•Brd2a is necessary for normal expression of patterning genes in anterior hindbrain•Deficiency of Brd2a leads to mispaired interneurons and excess kidney progenitors•Brd2 likely has an evolutionarily conserved role in patterning of segmented tissues
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