Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions

Abstract

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

Copper signalling: causes and consequences

“…The extremely diverse copper speciation may be represented by a collection of copper bioligands including small ions and molecules such as sulfide ion, amino acids like His, Cys, Met, Asp, Tyr, Thr, Gly; neurotransmitters such as ATP, norepinephrine [ 111 ]; γ-aminobutyric acid (GABA) [ 112 ], and constituents of dense core vesicle cargo neurotrophins ([ 113 ] and references cited) inositol phosphates (IPs) [ 114 ], low-density lipoproteins (LDL) [ 115 ]. Redox propensity of chelates between copper and pertinent peptides (tripeptide glutathione (γ-L-glutamyl-L-cysteinylglycine: GSH) [ 116 , 117 ]; peptide fragments of matricellular calcium-binding glycoprotein (secreted protein, acidic and rich in cysteine: SPARC) Gly-His-Lys (GHK) (for a recent review see [ 118 ] and proteins (metallothionein, ceruloplasmin, albumin, macroglobulin, transcuprein [ 3 , 19 , 119 – 122 ]), prion protein PrP C [ 65 ], amylin [ 123 ]) may present specific feature of transport and storage of copper. Likewise, many cuproproteins with redox, or redox-with-transport functions (mono-, di-, tetranuclear cupredoxins nitrite reductase, laccase, Cu, Zn-SOD1, amine oxidase CuAO, galactose oxidase, hemocyanin, tyrosinase, catechol oxidase, COX, N 2 O reductase, menaquinol NO reductase et cetera) [ 47 ], copper-transporting ATPases (Cu-ATPases, ATP7A and ATP7B) [ 124 – 126 ], divalent metal transporter DMT1 [ 127 ], copper transporters and chaperons Ctr1, Ctr2, Atox1 and CCS [ 128 , 129 ], diverse group of bacterial periplasmic copper binding proteins (CopC) [ 130 ] are known.…”

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells

“…The toxicity of the different preparation of nanoparticles (ECNs or DPPC:POPG(7:3)/ECNs) was measured through the determination of cell viability using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as previously reported 60 .…”

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine

“…Cell viability of RAW 264.7 plated in 48-well plates (2.5 × 10 5 cells/well) under our different experimental conditions was measured by the MTT assay as previously described [35,36]. Briefly, after the stimulation process, in the absence or in the presence of carnosine, MTT solution (1 mg/mL in DMEM medium) was added to each well followed by an incubation for 2 h at 37 • C. The formed crystals were melted with DMSO and used to read the absorbance at 569 nm using a microplate reader (Molecular Devices, Spectra Max M5, Sunnyvale, CA, USA).…”