Disrupted protein folding or decreased protein stability can lead to the accumulation of (partially) un-or misfolded proteins, which ultimately cause the formation of protein aggregates. Much of the interest in protein aggregation is associated with its involvement in a wide range of human diseases and the challenges it poses for large-scale biopharmaceutical manufacturing and formulation of therapeutic proteins and peptides. On the other hand, protein aggregates can also be functional, as observed in nature, which triggered its use in the development of biomaterials or therapeutics as well as for the improvement of food characteristics. Thus, unmasking the various steps involved in protein aggregation is critical to obtain a better understanding of the underlying mechanism of amyloid formation. This knowledge will allow a more tailored development of diagnostic methods and treatments for amyloid-associated diseases, as well as applications in the fields of new (bio)materials, food technology and therapeutics. However, the complex and dynamic nature of the aggregation process makes the study of protein aggregation challenging. To provide guidance on how to analyze protein aggregation, in this review we summarize the most commonly investigated aspects of protein aggregation with some popular corresponding methods.

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Aggregation can be selectively induced by aggregation-prone regions (APRs) contained in the target proteins.Aggregation inducing antimicrobial peptides (Pept-ins) contain homologous sequences to APRs of target proteins and exert their bactericidal effect by causing aggregation of a large number of proteins. To better understand the mechanism of action of Pept-ins and the resistance mechanisms, we analysed the phenotypic, lipidomic, transcriptomic as well as genotypic changes of laboratory derived Pept-in-resistant E. coli mutator cells. The analysis showed that Pept-in resistance mechanism is dominated by a decreased Pept-in uptake, both in laboratory derived mutator cells and clinical isolates. Our data indicates that Pept-in uptake involves the electrostatic attraction between Pept-in and bacterial membrane and follows a complex mechanism potentially involving many transporters. Furthermore, it seems more challenging for bacteria to become resistant towards Pept-ins which are less dependent on electrostatic attraction for uptake, suggesting future Pept-ins should be selected for this property.