Checking for reversibility of aggregation of UV-irradiated glycogen phosphorylase b under crowding conditions

https://doi.org/10.1016/j.ijbiomac.2016.02.010Get rights and content

Highlights

  • UV-irradiated Phb (UV⿿Phb) is a convenient target protein for the study of the initial stages of aggregation process.

  • UV⿿Phb aggregation induced by crowders at 10 °C can be reversed by dilution or by addition of Arg and Pro.

  • At 37 °C Arg induces disintegration of protein aggregates only at early stages of the aggregation process.

  • Proposed scheme of aggregation includes the formation of reversible, completely and partly dissociable aggregates.

Abstract

It is believed that the initial stages of protein aggregation are reversible and can be reversed by simple dilution, whereas prolonged exposure to factors responsible for denaturing proteins (for example, to elevated temperatures) results in the formation of irreversible aggregates. A new approach has been developed to discriminate the stage of the formation of reversible aggregates. Aggregation of UV-irradiated glycogen phosphorylase b (UV⿿Phb) was studied at 10, 25 and 37 °C in the presence of crowders (polyethylene glycol and Ficoll-70) using dynamic light scattering and analytical ultracentrifugation (pH 6.8; 0.1 M NaCl). The dilution of the protein solution in the course of aggregation at 10 °C results in the breakdown of protein aggregates suggesting that the aggregation process is reversible. When aggregation of UV⿿Phb is studied at 37 °C, reversibility is lacking. Chemical chaperones (arginine, proline) induce the breakdown of protein aggregates of UV⿿Phb formed at 10 °C. In the experiments carried out at 37 °C in the presence of crowder the addition of arginine results in disintegration of protein aggregates only at early stages of the aggregation process. It is assumed that general pathway of protein aggregation includes the formation of reversible, completely dissociable, partly dissociable and irreversible aggregates.

Introduction

Investigations of processes of protein aggregation are of great importance to biotechnologists involved with obtaining and storage of protein preparations [1], [2], [3] and also to biochemists studying the mechanisms of development of diseases associated with accumulation of protein aggregates [4], [5].

Denaturation of proteins induced by the action of different physical and chemical factors results in the formation of the unfolded states which reveal a high propensity to aggregation. Unfolded monomers may reversibly self associate to form soluble aggregates. Soluble aggregates may undergo internal structural rearrangement into aggregates where their constituent monomers are tightly bound to one another. Thus, initially formed reversible aggregates may be transformed into irreversible aggregates when time interval selected for registration of protein aggregation is sufficiently long [6], [7], [8]. In addition to reversible aggregates that exist in equilibrium with monomeric species and irreversible aggregates which when formed cannot be dissociated short of the addition of denaturants or reducing agents Narhi et al. [9], [10] developed the notion of dissociable aggregates. Though such aggregates are not reversible, it may still be possible to recover the monomeric species through application of heat, buffers or other conditions beyond a simple reversal of the conditions that led to aggregation. The detailed study of reversible and dissociable aggregates is made difficult by the fact that they are transiently accumulated during the aggregation process eventually forming irreversible aggregates.

Test systems designed for testing the agents possessing anti-aggregation activity and based on aggregation of proteins denatured by ultraviolet irradiation [11], [12], [13], [14] is a convenient tool for studying the initial stages of protein aggregation. Target protein denatured by UV-irradiation remains in non-aggregated state at low temperatures (about 10 °C), but reveals a high propensity to aggregation at physiological temperatures. Owing to this circumstance, the experiments with aggregation of UV-irradiated proteins at 37 °C can be used for estimation of the anti-aggregation activity of protein chaperones (for example, small heat shock proteins) and low-molecular-weight chemical chaperones (amino acids, cyclodextrins and so on) [12], [13], [14]. In contrast to test systems based on heat-induced aggregation of intact proteins, such experiments allow characterizing effect of the agents under testing directly on the stage of aggregation of denatured protein molecules (see also [15]). When studying aggregation stability of glycogen phosphorylase b denatured by ultraviolet (UV⿿Phb), it was shown that UV⿿Phb was relatively stable at 20 °C but was capable of forming large-sized aggregates under crowding conditions created by polyethylene glycol (PEG), trimethylamine N-oxide (TMAO) or Ficoll-70 [12]. Such an effect of crowding agents is consistent with the idea that crowding favors protein-protein interactions [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]. Special experiments should be performed to elucidate whether or not aggregation of UV-irradiated target protein under crowding conditions is reversible.

The goal of the present work was to study aggregation of UV⿿Phb in the presence of crowding agents at different temperatures (10, 25 and 37 °C) using dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) and to clarify the possibility of disintegration of protein aggregates by dilution of the solution or by the addition of chemical chaperones in the course of aggregation. Arginine (Arg) and proline (Pro) were used as chemical chaperones possessing the ability to suppress protein aggregation. Protection against protein aggregation by Arg [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38] and Pro [14], [35], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50] has been studied by many investigators.

It was shown that aggregates of UV⿿Phb formed at 10 °C in the presence of crowding agents could be disintegrated by dilution of the protein solution or by addition of 0.5 M Arg (or 1 M Pro). When aggregation of UV⿿Phb was studied at 37 °C in the presence of crowding agents, there was no disintegration of protein aggregates upon dilution; the breakdown of protein aggregates was observed only after addition of Arg at early stages of the aggregation process.

Section snippets

Materials

Hepes, l-arginine monohydrochloride, l-proline, glucose 1-phosphate, AMP and Ficoll with molecular mass of 70,000 Da (Ficoll-70) were purchased from ⿿Sigma⿿ (USA), NaCl was purchased from ⿿Reakhim⿿ (Russia), polyethylene glycol with molecular mass of 20,000 Da (PEG) was purchased from Ferak Berlin (Germany), glycogen was purchased from ⿿Olaine⿿ (Latvia). All solutions for the experiments were prepared using deionized water obtained with the Easy-Pure II RF system (Barnstead, USA).

Isolation of Phb and enzyme assay

Phb was purified

UV⿿Phb characteristics

On the basis of analysis of the enzymatic activity versus dose plots the dose of irradiation by UV light was chosen so that the residual activity of Phb was 1⿿3%. This dose was 9.4 J cm⿿2. According to DSC data, Phb irradiated by such a dose completely loses the native structure. DLS and AUC were used to characterize protein particles existing in the solution of UV⿿Phb. The size particle distribution for UV⿿Phb (1.5 mg mL⿿1) obtained by DLS at 10 °C was monomodal with average hydrodynamic radius Rh = 

Discussion

The formation of aggregates which are capable of dissociating into smaller species proceeds at the early stages of the process of aggregation of unfolded proteins. Reversible protein aggregation involves the stage of nucleation and stage of growth of aggregates [64], [65], [66]. The stage of nucleation is thermodynamically unfavorable (the change of free energy is positive) because of the loss in configurational entropy. When the aggregating phase contains a sufficient number of stable nuclei,

Conclusions

A growing body of scientific evidence indicates that the species formed early in the aggregation process, rather than mature amyloid fibrils, are the origins of pathological behavior in neurodegenerative conditions by inducing cell death [72], [73], [74], [75]. Valuable information on different types of species within the complex process of amyloid fibrillation can be obtained from disaggregation experiments [76], [77], [78], [79]. When studying aggregation and disaggregation of amyloid

Acknowledgments

This study was funded by the Russian Foundation for Basic Research (grants 16-04-00560-a and 14-04-01530-a) and the Program ⿿Molecular and Cell Biology⿿ of the Presidium of the Russian Academy of Sciences. The authors thank Dr. S. Yu. Kleymenov for carrying out DSC experiments.

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