Does the Crowded Cell-like Environment Reduce the Chaperone-like Activity of α-Crystallin?

Abstract

The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.

Concentration dependence of chaperone-like activities of α-crystallin, αB-crystallin and proline

“…The summary UV radiation dose obtained by Phb was equal to 13.5 J/cm 2 . Previously we showed that, judging from the measurements of the enzymatic activity and the data on differential scanning calorimetry, Phb completely lost enzymatic activity and native structure under these conditions of irradiation [18]. The samples of UV-irradiated Phb were centrifuged at 6800 × g for 5 min (10 • C) using a 5804R Eppendorf centrifuge.…”

“…The initial parts of the aggregation curves were analyzed by the following equation proposed by us earlier [18]:…”

“…Previously we demonstrated that ␣-crystallin suppressed aggregation of UV-irradiated Phb [18]. To have a quantitative estimation of the antiaggregation activity of ␣-crystallin, we analyzed the initial parts of the kinetic curves of aggregation obtained at various concentrations of ␣-crystallin.…”

“…To overcome the above-mentioned drawback of aggregation test systems, we proposed a new test system based on aggregation of UV-irradiated protein substrate (see [18]). In this work we showed that UV irradiation of Phb resulted in denaturation and inactivation of the enzyme molecules.…”

Dissociative mechanism for irreversible thermal denaturation of oligomeric proteins

“…Such reactions include protein isomerization, folding/unfolding and prot e i n -p r o t e i n i n t e r a c t i o n s ( C h e b o t a r e v a 2 0 0 7 ; Chebotareva et al 2001Chebotareva et al , 2004Chebotareva et al , 2010Chebotareva et al , 2013Chebotareva et al , 2015aEllis 2001;Ellis and Minton 2003;Gruebele et al 2016;Hall and Minton 2003;Kurganov and Chebotareva 2013;Kuznetsova et al 2014;Minton 2001;Roman et al 2011Roman et al , 2016Sluchanko et al 2015;Wills and Winzor 2011;Zhou et al 2008). Macromolecules (proteins, polysaccharides, neutral polymers) as well as low-molecular-weight compounds (for example, osmolytes) can be used in model experiments as crowding agents (Chebotareva et al 2001(Chebotareva et al , 2004Davis-Searles et al 2001;Ellis 2001;Jiao et al 2010;Roman et al 2011;Sluchanko et al 2015).…”