Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions.
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.
Department of Chemistry and Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
Giuseppe
G
Caruso
Division of Medical Biochemistry, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.
Donatella A
DA
Distefano
Division of Medical Biochemistry, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.
Paolo
P
Parlascino
Department of Chemistry and Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
Claudia G
CG
Fresta
Division of Medical Biochemistry, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy. lazzarig@unict.it.
Giuseppe
http://orcid.org/0000-0002-5917-7279
G
Lazzarino
Department of Chemistry and Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
Susan M
SM
Lunte
Division of Medical Biochemistry, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.
Vincenzo G
VG
Nicoletti
D000682:Amyloid / Q000633:toxicity*; D000818:Animals; D002460:Cell Line; D003300:Copper / Q000633:toxicity*; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000633:toxicity*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D000067759:Receptor for Advanced Glycation End Products / Q000235:genetics / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*
C000593871:AGER protein, human; D000682:Amyloid; D058228:Islet Amyloid Polypeptide; C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D000067759:Receptor for Advanced Glycation End Products; D017475:Receptors, Nerve Growth Factor; D003300:Copper
Aggregation; Amylin; Cell viability Copper Receptor-mediated cell toxicity; Oligomers