pH-responsive modulation of insulin aggregation and structural transformation of the aggregates.

Over the past two decades, much information has appeared on electrostatically driven molecular mechanisms of protein self-assembly and formation of aggregates of different morphology, varying from soluble amorphous structures to highly-ordered amyloid-like fibrils. Protein aggregation represents a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. This has awakened interest in identification of pH-triggered regulators of transformation of aggregation-prone proteins into structures of higher order.

Can aggregation of insulin govern its fate in the intestine? Implications for oral delivery of the drug.

The objective of this study is to elucidate the role of low-molecular weight biogenic agents, resembling dietary-derived products naturally occurring in the intestine, in the regulation of transformations of soluble aggregation-prone insulin into aggregates of higher order. In the course of model experiments, a striking potential of the amino acids L-arginine (Arg) and L-lysine (Lys) and a number of positively charged peptides to induce formation of heterogenic supramolecular structures of insulin was demonstrated under environment conditions where the protein aggregation in their absence was not observed. This phenomenon is assumed to be essential for elaboration of strategies of oral delivery of insulin to diabetic patients supplemented by controlling the pH values of the intestinal environment where the drug is released.

L-arginine induces protein aggregation and transformation of supramolecular structures of the aggregates.

Protein misfolding, self-assembly, and aggregation are an essential problem in cell biology, biotechnology, and biomedicine. The protein aggregates are very different morphologically varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils. The objective of this study was to elucidate the role of the amino acid L-arginine (Arg), a widely used suppressor of protein aggregation, in the regulation of transformations of soluble aggregation-prone proteins into supramolecular structures of higher order.

Transient transformation of oligomeric structure of alpha-crystallin during its chaperone action.

New evidence for dynamic behavior and flexible oligomeric structure of the molecular chaperone α-crystallin is presented. Based on the results of laser dynamic light scattering, centrifugal ultrafiltration, size exclusion chromatography, analytical ultracentrifugation and electrophoresis in polyacrylamide gel, addition of α-crystallin to fully reduced α-lactalbumin, used as a model protein substrate, at the stage of its start aggregate formation results in dissociation of multimeric structure of α-crystallin. In addition to large oligomers, transient low-sized assemblies are formed with the apparent molecular mass of 50-55 kDa that corresponds to the α-crystallin dimeric form associated with destabilized monomeric α-lactalbumin.

Formation of supramolecular structures of a native-like protein in the presence of amphiphilic peptides: Variations in aggregate morphology.

A striking potential of the amphiphilic dipeptides, Arg-Phe or Asp-Phe, to induce aggregation of a model protein, alcohol dehydrogenase in its native-like state, has been demonstrated under physiologically relevant conditions, using dynamic light scattering, fluorescence spectroscopy, circular dichroism, transmission electron- and atomic force microscopy. The peptide action resulted in accumulation of a variety of morphologically distinct supramolecular structures profoundly differing from those generated by the heat-induced aggregation at the early stages of the process, when amyloid fibril assemblies were not detectable. The biogenic amphiphilic agents are suggested to act as regulators of structural transformations of native-like proteins.

Paradoxical acceleration of dithiothreitol-induced aggregation of insulin in the presence of a chaperone.

The kinetics of dithiothreitol (DTT)-induced aggregation of human recombinant insulin and the effect of α-crystallin, a representative of the family of small heat shock proteins, on the aggregation process have been studied using dynamic light scattering technique. Analysis of the distribution of the particles by size measured in the course of aggregation showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with a hydrodynamic radius (R(h)) of about 90 nm. When studying the effect of α-crystallin on the rate of DTT-induced aggregation of insulin, it was demonstrated that low concentrations of α-crystallin dramatically accelerated the aggregation process, whereas high concentrations of α-crystallin suppressed insulin aggregation.

Mechanism of suppression of dithiothreitol-induced aggregation of bovine alpha-lactalbumin by alpha-crystallin.

The kinetics of dithiothreitol (DTT)-induced aggregation of alpha-lactalbumin from bovine milk has been studied using dynamic light-scattering technique. Analysis of the distribution of the particles formed in the solution of alpha-lactalbumin after the addition of DTT by size showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with the hydrodynamic radius (R(h)) of 80-100nm. Further growth of the protein aggregates proceeds as a result of sticking of the start aggregates.

Protein aggregates as depots for the release of biologically active compounds.

Protein misfolding and aggregation is one of the most serious problems in cell biology, molecular medicine, and biotechnology. Misfolded proteins interact with each other or with other proteins in non-productive or damaging ways. However, a new paradigm arises that protein aggregation may be exploited by nature to perform specific functions in different biological contexts.

Effects of arginine on kinetics of protein aggregation studied by dynamic laser light scattering and tubidimetry techniques.

Prevention of undesirable protein aggregation is an extremely important strategy in protein science, medicine, and biotechnology. Arginine is one of the most widely used low molecular weight solution additives effective in suppressing aggregation, assisting refolding of aggregated proteins, and enhancing the solubility of aggregation-prone unfolded molecules in vitro. However, the mechanism of suppression of protein aggregation by arginine is not well understood.

Chaperone-like activity of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor is a ubiquitous multifunctional cytokine having diverse immunological and neuroendocrine properties. Although this protein is known to be released into the circulation from the secretory granules of anterior pituitary or directly from immune cells as a consequence of stress, its participation in heat stress-induced aggregation of proteins has not yet been reported. We provide here the first evidence that the macrophage migration inhibitory factor possesses chaperone-like properties.