Effects of recombinant protein misfolding and aggregation on bacterial membranes

Abstract

The expression of recombinant proteins is known to induce a metabolic rearrangement in the host cell. We used aggregation-sensitive model systems to study the effects elicited in Escherichia coli cells by the aggregation of recombinant glutathione-S-transferase and its fusion with the green fluorescent protein that, according to the expression conditions, accumulate intracellularly as soluble protein, or soluble and insoluble aggregates. We show that the folding state of the recombinant protein and the complexity of the intracellular aggregates critically affect the cell response. Specifically, protein misfolding and aggregation induce changes in specific host proteins involved in lipid metabolism and oxidative stress, a reduction in the membrane permeability, as well as a rearrangement of its lipid composition. The temporal evolution of the host cell response and that of the aggregation process pointed out that the misfolded protein and soluble aggregates are responsible for the membrane modifications and the changes in the host protein levels. Interestingly, native recombinant protein and large insoluble aggregates do not seem to activate stress markers and membrane rearrangements.

Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

“…S6C), suggesting that the decrease in nitrocefin hydrolysis activity in the ⌬rsaF a F b strain is likely caused by internal RsaA accumulation. This result is consistent with recent findings that internal protein accumulation and aggregation can trigger decreased membrane permeability, altered membrane composition, and lipid arrangement in E. coli (56)(57)(58). Hence, RsaF a and RsaF b likely did not play a major role in providing outer membrane integrity to protect against toxicity of antimicrobials.…”

Protein aggregation in bacteria: the thin boundary between functionality and toxicity

“…A recent study provides the first experimental evidence that aggregation of recombinant cytoplasmic proteins affects directly the lipid moiety of bacterial membranes (Ami et al, 2009). It was shown that protein aggregation and misfolding induce changes, such as carbonylation or oxidation of host-specific proteins, a reduction in membrane permeability and rearrangements of its lipid components.…”

Fourier Transform Infrared Microspectroscopy as a Tool for Embryonic Stem Cell Studies

“…In this way, it is possible to study lipid phase transitions and changes in lipid composition (Casal & Mantsch, 1984). For instance, through the analysis of the changes in the CH2 and CH3 absorption bands, the stress response induced by protein aggregation in bacterial cells has been monitored in situ (Ami et al, 2009). Furthermore, through the CH stretching region analysis of mouse oocyte FTIR spectra and supported by the ester carbonyl band around 1740 cm -1 (see below), Wood and colleagues (Wood et al, 2008) found that lipids -whose composition within the oocytes drastically changes during maturation stages -could be considered potential markers of oocyte developmental competence.…”