Acceleration of protein aggregation by amphiphilic peptides: transformation of supramolecular structure of the aggregates.

Protein self-assembly and aggregation represent a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically, varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils, the latter being associated with molecular structures able to perform specific functions in living systems. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular-weight biogenic agents as regulators of transformation of aggregation-prone proteins into fibrillar structures. Short amphiphilic peptides can be considered for this role. Using dynamic light scattering, turbidimetry, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have demonstrated that the Arg-Phe dipeptide dramatically accelerates the aggregation of a model protein, α-lactalbumin, to generate morphologically different structures. TEM revealed transformation of spherical particles observed in the control samples into branched chains of fibril-like nanostructures in the presence of the peptide, suggesting that amphiphilic peptides can induce changes in the physicochemical properties of a protein substrate (net charge, hydrophobicity, and tendency to ß-structure formation) resulting in accumulation of peptide-protein complexes competent to self-assembly into supramolecular structures. A number of other short amphiphilic peptides have also been shown to accelerate the aggregation process, using alternative complementary protein substrates for identification of molecular recognition modules. Peptide-protein assemblies are suggested to play the role of building blocks for formation of supramolecular structures profoundly differing from those of the individual protein substrate in type, size, and shape.

Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins.

A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.

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.

Macrophage migration inhibitory factor: isolation from bovine brain.

The purification of macrophage migration inhibitory factor (MIF) from bovine brain cytosol and its partial characterization are reported. A rapid and relatively simple method for MIF isolation was developed based mainly on size-exclusion chromatography on Toyopearl TSK polymer having a tendency to adsorb MIF as compared to elution of other proteins with similar molecular weights. The method gives a high yield of MIF (0.1 mg homogenous protein per g wet tissue). The retardation is conveniently utilized to achieve good separations of MIF from other proteins of similar molecular weights. The isolated protein was identified as MIF by SDS-electrophoresis, immunoblotting, sequencing of the N-terminal amino acid residues, and also by determination of keto-enol tautomerase activity that is characteristic of MIF with p-hydroxyphenylpyruvic acid as a substrate.

Charge heterogeneity of bovine brain macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is known as a ubiquitous pluripotent cytokine originally identified for its capacity to inhibit the random migration of macrophages in vitro. It is recognized as an important regulator of the immunological, neuroendocrine and enzymatic processes. MIF is widely expressed in brain, but its role in the nervous system is not yet understood. In the course of the study of the primary structure of bovine brain MIF we have previously identified a number of MIF-related proteins having identical N-terminal sequences. In this paper we report the results of isoelectric focusing of MIF isolated to a homogeneous state from bovine brain that revealed MIF charge heterogeneity. We have detected isoelectric forms of MIF with pI values of 6.9, 7.0, 7.3, and 7.8. The diverse actions of MIF within the immuno-neuroendocrine system is suggested to be a result of its occurrence in different isoforms and oligomerization states.

Macrophage migration inhibitory factor: identification of the 30-kDa MIF-related protein in bovine brain.

Macrophage migration inhibitory factor (MIF) is a ubiquitous protein playing various immunologic, enzymatic, and hormonal roles. MIF was originally identified for its capacity to inhibit the random movement of macrophages in vitro. MIF is widely expressed in many tissues with particularly high levels in the nervous system. Using the reversed-phase HPLC, N-terminal microsequence analysis, and database searching, we have identified in bovine brain several MIF-like proteins. According to mass spectral analysis, the molecular masses for three of them were determined as 12,369.2, 12,299.7, and 9,496.2 Da. In addition, we have identified another MIF-related protein (29,568.9 Da) by Western blotting using anti-MIF antibody raised to MIF (having an apparent molecular weight of 12 kDa) isolated to homogeneity from bovine brain cytosol. The modified purification procedure was mainly based on exclusion- and ion-exchange chromatography. Using p-hydroxyphenylpyruvic acid as a substrate, we have demonstrated tautomerase activity of the isolated MIF. The N-terminal sequences for all MIF-like proteins were found to be identical. Several other higher molecular weight putative MIF-related proteins were also revealed in the bovine brain cytosol extract. A multifunctional nature of MIF is suggested to be a result of its occurrence in different oligomerization states in a wide variety of tissues and cells.

Identification of macrophage migration inhibitory factor isoforms in bovine brain.

In the course of the study of the primary structures and molecular mechanisms of action of immunologically active compounds of the nervous system we have isolated from the soluble fraction of total bovine brain two heat-stable proteins. The purification procedure was mainly based on DEAE-Servacel ion-exchange chromatography and reversed-phase HPLC. The proteins were identified by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences for MIF1 and MIF2 were found to be identical. According to mass spectral analysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third peptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by the oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this protein. A comparatively simple purification procedure is presented that may be widely used for simultaneous isolation in one run of MIF isoforms.

Peptidyl-prolyl cis-trans isomerases: structure and functions.

Peptidyl-prolyl cis-trans isomerases (PPI) catalyze cis-trans isomerization of imide bonds in peptides and proteins. This review summarizes the literature on the structure and functions of PPIs, their involvement in protein folding, and organization of PPI-containing receptors and membrane channels. A possible role of several PPIs in distant interactions between cells is discussed.

The interaction of (1-4)-fragment of thymosin beta 4 with calmodulin-sensitive cAMP phosphodiesterase from hypothalamus.

Evidence was accumulated indicating that cyclic nucleotides are involved in regulation of growth, differentiation and function of lymphoid cells. It was previously shown that the N-fragment (1-4) of thymosin beta 4 (Ac-Ser-Asp-Lys-Pro-OH) inhibits in vivo the entry of cell populations into S-phase. In the course of the study of the interrelationship between the immune and neuroendocrine systems we have found that the tetrapeptide caused incomplete competitive inhibition of hypothalamic calmodulin (CaM)-dependent phosphodiesterase (PDE) stimulated by CaM. In the presence of the peptide, the 20-fold increase of the constant for PDE activation by CaM was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis. The value of the inhibition constant (Ki) amounted to 600 nM. In the absence of CaM, the peptide at saturating concentrations reduced the basal activity of PDE nearly 2- to 3-fold. The effect of the peptide on PDE was noncompetitive with respect to cAMP. The results support our suggestion that the tetrapeptide realizes its effects in the immuno-neuroendocrine system by the mechanism of cyclic nucleotide metabolism.

[Inhibition of rat heart and brain cyclic adenosine-3',5'-monophosphate phosphodiesterase in vitro under the influence of neurohormone C]. / Ingibirovanie fosfodiésterazy tsiklicheskogo adenozin-3',5'-monofosfata serdtsa i mozga krys pod deistviem neirogormona C in vitro.

The work was devoted to one of the new neurohormones (neurohormone "C") obtained from the bovine hypothalamus which produced a pronounced relaxation of the coronary vessels. A study of the effect of "C" on cyclic 3',5'-adenosine monophosphate (cAMP) phosphodiesterase (PDE) showed "C" to be a potent inhibitor of PDE in the crude PDE-preparations of the rat heart and brain. Experiments were carried out on the 2000 g supernatant obtained from the homogenized tissues. It is supposed that the relaxant effect of "C" on the coronary vessels is due to its inhibitory action on PDE mediated by the accumulated cAMP.

[Inhibition of rat heart and brain cyclic 3',5'-adenosine monophosphate phosphodiesterase under the influence of neurohormone "C"]. / Ingibirovanie fosfodiésterazy tsiklicheskogo 3', 5'-adenozinmonofosfata serdtsa i mozga krys pod dei stviem neirogormona "C".

In vitro studies of the effect of neurohormone C on cyclic AMP phosphodiesterase activity of heart and brain showed that amounts of neurohormone equal to 5 ng inhibited significantly the activity of phos-phodiestherase in heart and even more strongly in brain. A comparison of the effect of the neurohormone with that of theophylline (10 mM) showed that very small amounts of the neurohormone brought about inhibition of enzyme activity that was equal to that observed from much larger concentrations of the theophylline. It is proposed that the inhibition of phosphodiestherase activity is of importance in the mechanism of the action of the neurohormone.