Glycation of Wild‐Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species

Abstract

Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type apomyoglobin have been also investigated.

Pinocembrin Protects from AGE-Induced Cytotoxicity and Inhibits Non-Enzymatic Glycation in Human Insulin

“…After 48 h of incubation with protein samples (40 μM), 2.5 × 10 5 cells were collected and resuspended in 500 μL of hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate, 50 μg/mL iodide propidium, RNAse A). Cells were incubated in the dark for 30 min, and samples were acquired on a FACSCalibur flow cytometer using Cell Quest software (Becton Dickinson, San Jose, CA, USA) and ModFitLT version 3 software (Verity Software House, Topsham, ME) as previously reported [45].…”

Vanillin Affects Amyloid Aggregation and Non-Enzymatic Glycation in Human Insulin

“…Cell viability was assessed as the inhibition of the ability of cells to reduce the metabolic dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product 90 . After indicated times of incubation with protein samples, cells were rinsed with phosphate buffer solution (PBS).…”

Glycation in Demetalated Superoxide Dismutase 1 Prevents Amyloid Aggregation and Produces Cytotoxic Ages Adducts

“…Recently, much attention has been devoted to the role played by glycation in stimulating amyloid aggregation and cellular toxicity. Results obtained for different protein models indicate that, depending on the protein, glycation can both promote amyloid aggregation or induce formation of oligomeric species, stabilized by covalent cross-links AGE-derived, that do not evolve into amyloid aggregates (Bouma et al, 2003 ; Lee et al, 2009 ; Rondeau et al, 2010 ; Oliveira et al, 2011 ; Iannuzzi et al, 2013a , 2014 , 2015 ; Adrover et al, 2014 ). Also, glycation can affect structural and physicochemical features of amyloid oligomers as well as their interaction to the cell membrane and subsequently modulate and/or induce the cell toxicity.…”