Abstract:Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β-transition and fibril formation of the highly amyloidogenic region spanning residues … Show more
“…In a cellular environment, membrane lipid compositions as well as extracellular matrix components are thought to mediate formation and deposition of amyloid fibrils in many amyloidogenic proteins 59 – 61 . Indeed, glycosaminoglycans such as heparin and heparan sulfate were shown to play critical roles in formation and cellular interaction of apoA-I amyloid fibrils 26 , 62 – 64 . In addition, it has been demonstrated that membrane binding strongly inhibits fibril formation by the N-terminal fragments of apoA-I through entrapping the protein in a stable α-helical structure 27 , 28 .…”
Section: Discussionmentioning
confidence: 99%
“…Alternatively, it is known that stabilization of the α-helical structure of proteins generates a kinetic trap in transition to β-strand structure, thereby inhibiting the aggregation and fibril formation 21 – 23 . In apoA-I, the N-terminal amino acid 1‒83 or 1‒93 fragments that are predominantly a random coil structure in solution have a strong propensity to form amyloid fibrils 11 , 24 – 26 . However, a lipid environment promotes the transition to the α-helical structure, thereby preventing the β-aggregation and fibril formation of the proteins 27 , 28 .…”
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.
“…In a cellular environment, membrane lipid compositions as well as extracellular matrix components are thought to mediate formation and deposition of amyloid fibrils in many amyloidogenic proteins 59 – 61 . Indeed, glycosaminoglycans such as heparin and heparan sulfate were shown to play critical roles in formation and cellular interaction of apoA-I amyloid fibrils 26 , 62 – 64 . In addition, it has been demonstrated that membrane binding strongly inhibits fibril formation by the N-terminal fragments of apoA-I through entrapping the protein in a stable α-helical structure 27 , 28 .…”
Section: Discussionmentioning
confidence: 99%
“…Alternatively, it is known that stabilization of the α-helical structure of proteins generates a kinetic trap in transition to β-strand structure, thereby inhibiting the aggregation and fibril formation 21 – 23 . In apoA-I, the N-terminal amino acid 1‒83 or 1‒93 fragments that are predominantly a random coil structure in solution have a strong propensity to form amyloid fibrils 11 , 24 – 26 . However, a lipid environment promotes the transition to the α-helical structure, thereby preventing the β-aggregation and fibril formation of the proteins 27 , 28 .…”
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.
“…The W50R mutation, known as an amyloidogenic mutation in apoA-I 57, 58 , places the strongly basic arginine residue into the nonpolar face of the amphipathic α-helix in residues 44–65 (Fig. 5C left), leading to destabilization of the helical structure.…”
Section: Resultsmentioning
confidence: 99%
“…S7). The apoA-I 44–65 peptide was synthesized by the solid-phase method with Fmoc chemistry 58, 60 . These apoA-I variants and peptide were dialyzed from 6 M guanidine hydrochloride solution into PBS before use.…”
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.
“…Nevertheless, the disruptive presence of a positive charge in replacement of the aromatic Trp is detected in our experimental design by an increased yield in the Bis-ANS binding and minor change in the near UV spectra, together with the detection of stronger binding to heparin at acidic pH. It is worth mentioning that the N-terminal 1–83 fragment of W50R variant was shown to participate in heparin-mediated fibril formation [39]. Fig 9. Locations of amyloidogenic mutations W50R and L60R in the structure of apoA-IThe structure was obtained by the PyMOL Molecular Graphics System, Version 2.0 (Schrödinger, LLC), from the X-ray crystal structure of Δ(185-243) apoA-I (PDB ID 3R2P).…”
23Since the early description of different human apolipoprotein A-I variants 24 associated to amyloidosis, the reason that determines its deposition inducing organ 25 failure has been under research. To shed light into the events associated to protein 26 aggregation, we studied the effect of the structural perturbations induced by the 27 replacement of a Leucine in position 60 by an Arginine as it occurs in the natural 28 amyloidogenic variant (L60R). Circular dichroism, intrinsic fluorescence measurements 29 and assays of binding to ligands indicate that L60R is more unstable, more sensitive to 30 proteolysis and interacts with sodium dodecyl sulfate (a model of negative lipids) more 31 than the protein with the native sequence and other natural variant tested, involving a 32 replacement of a Trytophan by and Arginine in the amino acid 50 (W50R). In addition, 33 the small structural rearrangement observed under physiological pH leads to the release 34 of tumor necrosis factor α and interleukin-1β from a model of macrophages. Our results 35 strongly suggest that the chronic disease may be a consequence of the loss in the native 36 conformation which alters the equilibrium among native and cytotoxic proteins 37 conformation. 38 39 40 41 42 Human apolipoprotein A-I (apoA-I) is the main protein associated to high 43 density lipoproteins (HDL). It is synthesized in liver and intestine and both the liver and 44 the kidney are the major sites of its catabolism. Its multiple functions as lipid transport, 45 endothelial homeostasis and inhibition of inflammatory pathways [1][2][3] could 46 however be counter balanced by the presence of single point mutations which could, by 47 not yet completely known pathways, induce its misfunction, increased clearance rate or 48 its tendency to aggregate [4][5]. Hereditary apoA-I amyloidosis has been described 49 since 1969, and is characterized by specific deposits of natural variant proteins within 50 organs, in a pattern which is dependent on the mutation in the protein sequence. From 51 the more than 20 known natural variants, almost half result from substitutions between 52 amino acids 26 and 107, and involve with different severity hepatic and kidney failure.53 Instead, a "hot spot" affecting residues 173-178 was described, in which variants are 54 especially associated to cardiac, skin and testis damage. The reasons for the different 55 deposit patterns are still unknown but may be the consequence of long term seeding of 56 misfolded proteins that yield a final fibrillar conformation. 57 The first identified apoA-I natural mutant was the result of a substitution of a 58 Glycine by an Arginine in position 26 from the native sequence (Gly26Arg, in the 59 abbreviated nomenclature G26R), inducing in patients peripheral neuropathy, peptic 60 ulcer, and nephropathy [6]. The next mutations described in the N terminus of apoA-I 61 were Trp50Arg (W50R) [7] and Leu60Arg (L60R) [8]. These variants show similarities 62 with G26R: in each, a neutral residue is replaced by an Arginine thu...
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