Why and how protein aggregation has to be studied in vivo

Abstract

The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid aggregation in several neurodegenerative and systemic diseases. To this goal, it became more and more evident the crucial relevance of studying protein aggregation in the complex cellular environment, since it allows to take into account the cellular components affecting protein aggregation, such as chaperones, proteases, and molecular crowding. Here, we discuss the use of several biochemical and biophysical approaches that can be employed to monitor protein aggregation within intact cells, focusing in particular on bacteria that are widely employed as microbial cell factories.

Twin arginine translocation system in secretory expression of recombinant human growth hormone

“…Lack of eukaryotic post translational modifications and inclusion body formation are the main disadvantages. The improper conformation could make the expressed proteins undergo proteolytic degradation or aggregation (3). Intra- or inter-molecular disulfides bonds cannot be easily formed in the reducing cytoplasm of E. coli which facilitates aggregation of certain disulfide bond–rich proteins (4).…”

Synthesis and Multitarget Biological Profiling of a Novel Family of Rhein Derivatives As Disease-Modifying Anti-Alzheimer Agents

“…Because IBs contain highly ordered amyloid-like structures and their formation seems to share mechanistic features with amyloid self-assembly, they have been recently proposed as a model to study amyloid aggregation. 72,73 We have recently developed a methodology that allows a fast, simple, and inexpensive evaluation of the anti-aggregating activity of putative inhibitors, which is based on the in vivo staining with thioflavin S of IBs in intact E. coli cells that overexpress a given amyloidogenic protein in the presence and absence of the inhibitors, and monitoring of the corresponding changes in the fluorescence of thioflavin S. 74 The applicability of this method to the screening of both Aβ42 and tau aggregation inhibitors has been recently demonstrated. 74 Worthy of note, the Aβ42 anti-aggregating activities determined through this method for a number of known active and inactive inhibitors were very similar to those previously reported in in vitro assays using synthetic peptides, thereby validating this methodology.…”

Thioflavin-S Staining of Bacterial Inclusion Bodies for the Fast, Simple, and Inexpensive Screening of Amyloid Aggregation Inhibitors

“…Indeed, in living organisms proteins are prone to associate and interact with other cellular components, giving rise to complex interaction networks [23,24] that are absent in in vitro tests [25]. Thus, in vivo A aggregation is dependent on a number of factors that define its complex cellular environment [1,26,27], which cannot be captured in in vitro assays. Indeed, it has been suggested that A aggregation can proceed through different pathways in vitro and in vivo thereby leading to different A aggregates [12].…”

“…In this light, bacteria have emerged as suitable models to monitor protein aggregation [27,28]. Protein aggregation also occurs during the production of heterologous proteins in prokaryotic systems, giving rise to insoluble protein aggregates, the so-called inclusion bodies (IBs), which limits the application of bacteria for recombinant protein production in the biotechnology industry [29].…”

“…Therefore, it seems that the establishment of an inter-backbone, hydrogen-bonded network that stabilizes related fibrillar structures enriched in the β-sheet conformation is a common force driving protein aggregation in the cell. Different methodologies can be used to monitor protein aggregation in bacteria, which involve the fluorescence detection of either genetically encoded fusion tags such as the green fluorescent protein (GFP) or conformational-sensitive fluorescent dyes, prominently thioflavin-S (Th-S) [27]. Regarding the first methodology, we have very recently developed a new method for the detection of inhibitors of metal-promoted A42 aggregation using purified bacterial IBs formed by an A42-GFP fusion protein in Escherichia coli [34].…”