Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity

Abstract

The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.

Intermolecular interactions in highly concentrated protein solutions upon compression and the role of the solvent

“…[1][2][3][4] Knowledge of protein-protein interactions in condensed fluid phases is also crucial for understanding the functional and structural stability of proteins and to yield molecular insights into processes such as protein crystallization, aggregation, and fibrillation. [5][6][7][8][9][10][11] The latter involves diseases that occur due to undesired protein nucleation and aggregate formation. Classic examples are formation of polymer fibers of sickle hemoglobin molecules within the red blood cells that produces sickle cell anemia, age-related cataracts produced by the undesired aggregation of γ -crystallin, or the formation of amyloid fibres via cross-β-sheet formation in a series of debilitating diseases such as Alzheimer's, Parkinson's, Creutzfeld-Jakob disease, or diabetes mellitus type 2.…”

Low crowding agent concentration destabilizes against pressure unfolding

“…Seelinger et al studied the fibrillation of the type-2 diabetes mellitus related human islet amyloid polypeptide. Stabilization of the monomeric form and consequently suppression of the fibrillation was found [7]. On the contrary the addition of high concentrations of different polymers dramatically accelerated alpha-synuclein fibrillation in vitro depending on the nature, length and concentration of the polymer [8].…”

“…These crowding agents can be hard particles like dextran or relatively open structures like Ficoll [13]. Another possibility is to use proteins (other than the test protein) [7]. The aim is to select a crowding agent, which does not have any specific interaction with the test protein, and allows measuring purely the excluded volume effect.…”

High Pressure Bioscience

“…Moreover, many examples involve the role of amyloid fibres via cross-"sheet formation in a series of debilitating diseases such as Alzheimer's, Parkinson's, Creutzfeld-Jakob disease or diabetes mellitus type 2 (Grudzielanek et al 2006;Seeliger et al 2013;Silva et al 2014). Classic examples are the formation of polymer fibers of sickle hemoglobin molecules within the red blood cells leading to sickle cell anemia, or age-related cataracts produced by the undesired aggregation of "-crystalline within the vitreous fluid of the eye.…”