How is protein aggregation investigated in vivo
Why and how protein aggregation needs to be studied in vivo
Understanding protein aggregation is a key issue in various areas of protein science, from heterologous protein production in biotechnology to amyloid aggregation in various neurodegenerative and systemic diseases. To this end, the critical relevance of studying protein aggregation in the complex cellular environment has become increasingly evident, as the cellular components that influence protein aggregation, such as chaperones, proteases, and molecular crowding, can be taken into account. Here we discuss the use of various biochemical and biophysical approaches that can be used to monitor protein aggregation in intact cells, with a particular focus on bacteria, which are often used as factories for microbial cells.
Protein aggregation is a relevant process in various areas of biomedicine and biotechnology. Indeed, many diseases are associated with the deposition of amyloid aggregates , while the formation of inclusion bodies (IBs) often occurs during the production of heterologous proteins [2, 3]. In particular, bacterial IBs, which have long been viewed as a bottleneck in the production of recombinant proteins, have recently gained attention as a valuable source of active recombinant proteins [6–8] and as a model system for amyloid studies [4, 5] [ 9-15]. In addition, the special structural properties of IBs and the observation that the aggregated proteins can retain their activity opened up the possibility of IBs in biocatalysis , in regenerative medicine  and in the controlled release of therapeutic polypeptides [ 18] to be used. 19].
The misfolding and aggregation of proteins has been studied extensively in the test tube, therefore under conditions that are far removed from the physiological and pathological conditions. For this reason, it is important to extend these studies to intact cells in order to take into account the complexity of the cellular environment, which plays a crucial role in the coordination of protein aggregation .
In this comment we have turned our attention to the various approaches that can be used to monitor protein aggregation in bacterial cells (Table 1). We should note that most of these approaches have been successfully used to monitor protein aggregation even in intact eukaryotic cells, including yeast and mammals.
Some of the most commonly used methods for studying protein aggregation in situ are based on the detection of fluorescence from genetically encoded fusion labels or from conformationally sensitive fluorescent dyes. In the first case, one of the most important tools is the green fluorescent protein (GFP) and its variants, like the yellow, blue and red, which are used to obtain fluorescent chimeric proteins that are easily detectable by fluorescence microscopy and flow cytometry.
This approach has been used, for example, to investigate the presence of functional proteins embedded in bacterial IBs [22–24]. Interestingly, it has been observed in recent work that the fusion of self-assembling or surfactant-like peptides with various proteins makes it possible to obtain active IBs, the formation of which has been demonstrated in vivo by monitoring the fluorescence of GFP - taken as a model system - fused to the peptide. Indeed, the bacterial cell images obtained by confocal microscopy showed diffuse fluorescence when GFP was expressed alone in a soluble form. If instead the GFP was expressed fused to the self-assembling or surfactant-like peptide, the fluorescence appeared to be localized in the cell, indicating the formation of active IBs [23, 24]. In addition, the use of the GFP tag as a reporter for the corrected folding was used to screen for Aβ mutations and chemical compounds that are able to tune the aggregation tendency of the peptide. In particular, it should be noted that the fluorescence of the fusion protein in intact cells correlates inversely with the aggregation of the Aβ-GFP fusion protein [25–27].
Notably, fluorescent protein fusion has also been used to study the mechanism of protein deposition at the single cell level  and the specificity of protein-protein interaction during in vivo protein deposition. For this purpose, Morell and colleagues carried out, for example, Förster resonance energy transfer (FRET) experiments in prokaryotic cells, whereby two self-assembling proteins, the Aβ42 amyloid peptide and the VP1 capsid protein, were labeled with suitable fluorescent protein variants . In this way, the specificity of protein deposition was indicated by a higher FRET efficiency observed when the two dyes were fused to the same polypeptide rather than the different ones.
Other applications based on fluorescence analysis to detect protein aggregation in vivo in real time include labeling the target protein with a tetra-cysteine sequence (Cys-Cys-XY-Cys-Cys) that specifically contains bis-arsenic fluorescein binds dye based on (FIAsH) . This intelligent approach enables the monitoring of the formation of hyperfluorescent aggregates in intact cells by simple detection of bulk cell fluorescence or by fluorescence microscopy [30, 31].
Protein aggregation can also be studied in vivo using conformation-sensitive dyes such as thioflavine-S (Th-S), the fluorescence spectroscopic features of which change upon interaction with amyloid aggregates. As recently reported in the literature, the ability of Th-S to be internalized in bacterial cells has been used to detect intracellular amyloid-like aggregates by fluorescence spectroscopy, microscopy, and flow cytometry. Interestingly, this approach may represent a new tool to study the effects of amyloid inhibitors in an intracellular environment .
Among the spectroscopic techniques that can be used to study protein aggregation in intact cells, Fourier Transform Infrared Spectroscopy (FTIR) has the advantage of being a label-free tool. In particular, the detection of protein aggregates is based on the presence of a specific marker band due to the formation of intermolecular β-sheet structures [33, 34]. Using this approach, it was possible to monitor the kinetics of IB formation in growing E. coli cells under various expression conditions . Interestingly, since the infrared response of an intact cell represents a chemical fingerprint of its main biomolecules , IR spectral analysis also enables complementary information to be obtained about cell processes associated with protein aggregation, including, for example, the effects on cell membranes .
In addition, the IR investigation of extracted IBs enables important information about the structural properties of the aggregated protein [34, 38, 39] and in particular the detection of native secondary structures of the proteins in IBs. For these reasons, the IR approach is a useful tool in identifying the best conditions that will make it possible to modulate not only the degree of protein aggregation but also the quality of the protein within the IBs.
More detailed structural information of the protein embedded in IBs can be obtained by nuclear magnetic resonance spectroscopy (NMR). This technique has not only been used to characterize isolated [40–42] IBs, but also IBs in cells . For example, in the pioneering work of Curtis-Fiske and colleagues, solid-state NMR was used to study whole bacterial cells that express the HA2 subunit of the influenza virus hemagglutinin protein in the form of IBs. In this way it was by labeling the carbonyl and nitrogen in the backbone ( 13 CO and 15 N) possible for each amino acid to identify the localization of native α-helices of the functional domain of the protein and also to reveal the protein conformational heterogeneity within IBs .
Finally, the assessment of protein aggregation in intact cells could also be approached by a biochemical approach based on the use of gene promoters that are specifically triggered by protein misfolding and aggregation [44–46]. For example, the expression of the β-galactosidase reporter under the control of the chaperone IbpB promoter, which was specifically activated by misfolded proteins, made it possible to estimate the protein aggregation accumulated in the cell . Using this approach, together with complementary biochemical and biophysical analyzes, the recombinant expression of glutathione-S-transferase and its fusion with GFP were investigated, the aggregation of which can be adjusted by changing the expression conditions. Interestingly, it was found that in this model system misfolded proteins and soluble aggregates - but not the soluble native protein or IBs - lead to a significant reorganization of cell membranes and host protein expression , a relevant result in the context of proteotoxicity.
We emphasize here the need to expand the study of protein aggregation in an intracellular environment in the presence of factors such as chaperones, proteases, and molecular densification, which can crucially influence the aggregation process in vivo.
Indeed, it will be necessary to complement studies in the test tube with those in intact cells, not only to gain a better understanding of the mechanisms underlying protein aggregation, but also to identify the factors that can modulate aggregation, such as B. the protein expression conditions. Mutations and the Effects of Chemical Compounds.
From this point of view, it will be highly desirable to further develop methods that could enable studies in intact cells, not only for a fundamental understanding of aggregation in situ, but also for applications in recombinant protein production and for the screening of compounds that inhibit aggregation, a relevant topic in medical therapies.
Bis-arsenic fluorescein based dye
Förster resonance energy transfer
Fourier transform infrared
Green fluorescent protein
Nuclear magnetic resonance
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