Accelerates Protein Aggregation Research Articles

Accelerated protein aggregation induced by macrophage migration inhibitory factor under heat stress conditions

Kinetics of thermal aggregation of model protein substrates (glycogen phosphorylase b from rabbit skeletal muscle and yeast alcohol dehydrogenase) were investigated under heat stress conditions (41-48 degrees C) in the presence of macrophage migration inhibitory factor (MIF), a heat-stable hydrophobic protein (12.5 kD). Anti-chaperone MIF activity found by turbidimetry manifests itself in significantly accelerated protein aggregation and increased limiting value of apparent optical absorption at 360 nm and t --> infinity in the sub-stoichiometric range of MIF concentrations. The aggregation kinetics is shown to have cooperative character. Possible reversibility of aggregation after removal of denaturing conditions was demonstrated using alcohol dehydrogenase aggregation at a temperature close to the physiological level (41.5 degrees C). This reversibility is caused by solubility of aggregates and stabilization of oligomeric structure of the substrate as a result of MIF binding to the partially denatured protein. The data suggest that in spite of distinct anti-chaperone effect, the chaperone-like activity of MIF can be observed in the case of heat stress removal and restoration of the system to normal conditions.

Mechanism for benzyl alcohol‐induced aggregation of recombinant human interleukin‐1 receptor antagonist in aqueous solution

Benzyl alcohol, an antimicrobial preservative, accelerates aggregation and precipitation of recombinant human interleukin‐1 receptor antagonist (rhIL‐1ra) in aqueous solution. The loss of native monomer during incubation at 37°C was determined by analysis of sample aliquots with size exclusion high performance liquid chromatography (SE‐HPLC). Benzyl alcohol caused minor perturbation of the tertiary structure of the protein without changing its secondary structure, documenting that the preservative caused a minor shift in the protein molecular population toward partially unfolded species. Consistent with this conclusion, in the presence of benzyl alcohol the rate of H‐D exchange was accelerated and the fluorescence of 1‐anilinonaphthalene‐8‐sulfonic acid in the presence of rhIL1ra was increased. Benzyl alcohol did not alter the free energy of unfolding based on unfolding experiments in urea or guanidine HCl. With differential scanning calorimetry it was determined that benzyl alcohol reduced the apparent Tm of rhIL‐1ra, but this effect occurred because the preservative lowered the temperature at which the protein aggregated during heating. Isothermal calorimetry documented that the interaction of benzyl alcohol with rhIL‐1ra is relatively weak and hydrophobically driven. Thus, benzyl alcohol accelerates protein aggregation by binding to the protein and favoring an increase in the level of partially unfolded, aggregation‐competent species. Sucrose partially inhibited benzyl alcohol‐induced aggregation and tertiary structural change. Sucrose is preferentially excluded from the surface of the protein, favoring most compact native state species over expanded aggregation‐prone forms. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:3076–3089, 2004

Tyrosine-to-cysteine modification of human alpha-synuclein enhances protein aggregation and cellular toxicity.

The deposition of alpha-synuclein and other cellular proteins in Lewy bodies in midbrain dopamine neurons is a pathological hallmark of Parkinson's disease. Nitrative and oxidative stress can induce alpha-synuclein protein aggregation, possibly initiated by the formation of stable cross-linking dimers. To determine whether enhanced dimer formation can accelerate protein aggregation and increase cellular toxicity, we have substituted cysteine for tyrosine at positions 39, 125, 133, and 136 in human wild-type (WT) alpha-synuclein, and in A53T and A30P mutant alpha-synuclein. To reduce the likelihood of cross-linking, phenylalanine was substituted for tyrosine at the same sites. We have found that overexpression of Y39C or Y125C mutant proteins leads to increased intracellular inclusions and apoptosis in a rat dopaminergic cell line (N27 cells) and in human embryonic kidney 293 cells. Expression of Y133C, Y136C, and all four Tyr-to-Phe mutations were not more cytotoxic than WT control. Exposure to oxidative stress increased Y39C and Y125C alpha-synuclein aggregation and toxicity. Dimers and oligomers were found in Triton X-100-soluble fractions from adenovirus-mediated overexpression of Y39C and Y125C in N27 cells. In contrast, WT beta-synuclein and all four Tyr-to-Cys mutant beta-synucleins did not cause protein aggregation and cell death. We conclude that cysteine substitution at critical positions in the alpha-synuclein molecule can increase dimer formation and accelerate protein aggregation and cellular toxicity of alpha-synuclein.