Zinc Determines Dynamical Properties and Aggregation Kinetics of Human Insulin

Abstract

<jats:title>Abstract</jats:title><jats:p>Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.</jats:p><jats:sec><jats:title>Statement of Significance</jats:title><jats:p>Localized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.</jats:p></jats:sec>

Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments

“…Fibrils and spherulites are both commonly formed under highly acidic conditions at elevated temperatures, and they can co-exist [17, 18]. This makes it difficult to prepare samples containing only fibrils or spherulites.…”

“…This makes it difficult to prepare samples containing only fibrils or spherulites. We investigated the effect of salt concentration (0 mM NaCl or 500 mM NaCl) and shaking speed (0 rpm or 600 rpm), which are parameters known to affect fibril or spherulite formation [4, 17, 20, 30]. Here, the parameters were investigated in a formulation consisting of 5 mg/mL human insulin in 20% acetic acid solution at pH 2.0 and the samples were stressed at 60 °C for 24 hours.…”

“…This is consistent with recent results by Pounot et al . [17] for human insulin in aqueous solutions with hydrochloric acid, where it was argued that the mechanical perturbation prevents formation of sufficiently stable nuclei during spherulite development. Fibrils formed both under shaking and quiescent conditions, where shaking produced shorter fibrils compared to fibrils formed at quiescent conditions (Fig.…”

“…Insulin is a biotherapeutic protein, often utilized for studying aggregation processes [6,15,16], due to its ability to form amyloid-like fibrils and spherulites at acidic pH and elevated temperatures [17,18]. Amyloid-like fibrils are elongated aggregates, with a section diameter of a few nanometres and length of several micro metres [19][20][21].…”

Strategies for Interference of Insulin Fibrillogenesis: Challenges and Advances

“…Inhibitory approaches broadly proposed as stable insulin formulations with added stabilizers and engineered insulin analogues with heat or mechanical stress withstanding property (Figure 1). Examples of external agents that halt insulin agglomeration are aromatic compounds, [44,45] bio-inspired peptides, [46] hybrid peptide conjugates, [47] nanoparticles, [48] trehalose, [49,50] polyphenols, [51] Zinc, [52] dyes, [53] metal complexes, [54][55][56] hydrophobic interfaces, [57] functionalized meso-porous silica, [58] etc. Another paradigm used to enhance insulin intrinsic stability is through insulin engineering which mainly involves amino acid substitutions, [59,60] halogenated insulin analogues, [61,62] intramolecular disulfide bond introduction, [63,64] replacement of natural disulfide bond with non-reducible cystathionine/cystine bond, [65] single chain insulin analogues, [66][67][68][69] glycosylated insulin analogues, [70] analogues inspired from insulin like venom of marine snails, [71] etc.…”