Fluorescence study of the effect of the oxidized phospholipids on amyloid fibril formation by the apolipoprotein A-I N-terminal fragment

The effects of the oxidized phospholipids (oxPLs) on amyloid fibril formation by the apolipoprotein A-I variant 1-83/G26R have been investigated using Thioflavin T fluorescence assay. All types of the PoxnoPC assemblies (dispersions, micelles and lipid bilayer vesicles) induced retardation of amyloid nucleation and elongation and the enhancement of the 1-83/G26R fibrillization, although PazePC micelles completely prevented protein aggregation at low protein-to-lipid molar ratios. The ability of PazePC to inhibit 1-83/G26R aggregation was explained by the protein-lipid electrostatic interactions, which either stabilize the a-helical structure of the membrane-associated 1-83/G26R or facilitate the protein solubilization by the detergent micelles. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

JRAB shifts “dancing style” of cell clusters

In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the “law and order” allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides “law and order” in collective cell migration

Effect of Phosphatidylserine and Cholesterol on Membrane-mediated Fibril Formation by the N-terminal Amyloidogenic Fragment of Apolipoprotein A-I

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles

Severe refractory TAFRO syndrome requiring continuous renal replacement therapy complicated with Trichosporon asahii infection in the lungs and myocardial infarction: an autopsy case report and literature review

Abstract Background TAFRO (thrombocytopenia, anasarca, fever, reticulin myelofibrosis/renal failure, and organomegaly) syndrome is a systemic inflammatory disorder and unique clinicopathological variant of idiopathic multicentric Castleman disease that was proposed in Japan. Prompt diagnosis is critical because TAFRO syndrome is a progressive and life threating disease. Some cases are refractory to immunosuppressive treatments. Renal impairment is frequently observed in patients with TAFRO syndrome, and some severe cases require hemodialysis. Histological evaluation is important to understand the pathophysiology of TAFRO syndrome. However, systemic histopathological evaluation through autopsy in TAFRO syndrome has been rarely reported previously. Case presentation A 46-year-old Japanese man with chief complaints of fever and abdominal distension was diagnosed with TAFRO syndrome through imaging studies, laboratory findings, and pathological findings on cervical lymph node and bone marrow biopsies. Interleukin (IL)-6 and vascular endothelial growth factor (VEGF) levels were remarkably elevated in both blood and ascites. Methylprednisolone (mPSL) pulse therapy was initiated on day 10, followed by combination therapy with PSL and cyclosporine A. However, the amount of ascites did not respond to the treatment. The patient became anuric, and continuous renal replacement therapy was initiated from day 50. However, the patient suddenly experienced cardiac arrest associated with myocardial infarction (MI) on the same day. Although the emergent percutaneous coronary intervention was successfully performed, the patient died on day 52, despite intensive care. Autopsy was performed to ascertain the cause of MI and to identify the histopathological characteristics of TAFRO syndrome. Conclusions Bacterial peritonitis, systemic cytomegalovirus infection, and Trichosporon asahii infection in the lungs were observed on autopsy. In addition, sepsis-related myocardial calcification was suspected. Management of infectious diseases is critical to reduce mortality in patients with TAFRO syndrome. Although the exact cause of MI could not be identified on autopsy, we considered embolization by fungal hyphae as a possible cause. Endothelial injury possibly caused by excessive secretion of IL-6 and VEGF contributed to renal impairment. Fibrotic changes in anterior mediastinal fat tissue could be a characteristic pathological finding in patients with TAFRO syndrome

The extreme N-terminal region of human apolipoprotein A-I has a strong propensity to form amyloid fibrils

AbstractThe N-terminal 1–83 residues of apolipoprotein A-I (apoA-I) have a strong propensity to form amyloid fibrils, in which the 46–59 segment was reported to aggregate to form amyloid-like fibrils. In this study, we demonstrated that a fragment peptide comprising the extreme N-terminal 1–43 residues strongly forms amyloid fibrils with a transition to β-sheet-rich structure, and that the G26R point mutation enhances the fibril formation of this segment. Our results suggest that in addition to the 46–59 segment, the extreme N-terminal region plays a crucial role in the development of amyloid fibrils by the N-terminal fragment of amyloidogenic apoA-I variants

Fluorescence Analysis of the Lipid Binding-Induced Conformational Change of Apolipoprotein E4

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix–helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL₃ suggests that not only apoE–lipid interactions but also protein–protein interactions are important for apoE4 binding to HDL₃