Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

Abstract

UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, alpha-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

Fig. 1

Expression and purification of DUNC-45, its suppression of α-lactalbumin or CS aggregation and its facilitation of CS refolding. (A) Purification of DUNC-45. The figure shows the results of SDS-PAGE analysis (10% polyacrylamide gel) of DUNC-45 levels in E. coli and during purification steps. 1) total cell lysate, 2) total soluble protein, 3) total insoluble protein, 4) run-through eluate from a nickel affinity column, 5) 5 mM imidazole wash, 6) 200 mM imidazole wash, 7) 500 mM imidazole elution (arrow indicates DUNC-45 band). (B) DTT-induced aggregation of α-lactalbumin in the absence or presence of DUNC-45. Curves 1 and 3 represent LS of α-lactalbumin at 37°C with and without DTT respectively. Curve 2 represents LS of α-lactalbumin at 37°C with DTT in the presence of DUNC-45. (C) Heat-induced aggregation of citrate synthase (CS) in the absence or presence of DUNC-45. Light scattering of 0.25 µM CS in 100 mM NaCl, 20 mM Tris-HCl, pH 7.5 in the absence (curve 1) or presence of 0.5 µM DUNC-45 (curve 2) was measured at 45°C. Curve 3 represents LS of DUNC-45 at 45°C. (D) Urea-induced unfolding of citrate synthase (CS) and its refolding facilitated by DUNC-45. Refolding of CS was carried out in the presence (curve 2) or absence (curve 1) of DUNC-45 at 25°C after 100 fold dilutions.

Fig. 2

Aggregation of myosin S-1 fragment as detected with light scattering in the absence or presence of DUNC-45. (A) Light scattering (LS) of Drosophila S-1 in the absence or presence of DUNC-45 or BSA. Curves 1 and 2 represent the LS of Drosophila S-1 (0.1 µM) in the presence or absence of BSA at 37°C respectively. Curves 3 and 4 represent the LS of Drosophila myosin S-1 in the presence of DUNC-45 (0.1 and 0.4 µM respectively) at 37°C. Curve 5 represents the LS of DUNC-45 (0.1 µM) at 37°C. (B) Aggregation of chicken S-1 in the absence (curve 1) or presence of DUNC-45 (curve 2) detected with LS. Aggregation conditions are similar to Drosophila S-1 except aggregation was carried out at 43°C. Curve 3 represents the LS of DUNC at 43°C.

Fig. 3

Light scattering and electron microscopy of HMM in the absence or presence of DUNC-45. (A). LS of chicken skeletal muscle myosin HMM fragment in the absence (curve 1) or presence of (curve 2) of DUNC-45. Curves 3 and 4 represent LS of HMM at 25°C in the presence or absence of DUNC-45. (B) Electron microscopy of HMM, in the absence and in the presence of DUNC-45. When HMM was incubated with DUNC-45 at 25°C the majority of the HMM molecules were well-preserved, two-headed, short-tailed structures (top row). DUNC-45 consistently appeared as a globular protein about the size of a single myosin S-1 head (red arrows, top row). When HMM was heated to 43°C for 30 min, most of the molecules collapsed and fused together to form intra- or inter-molecular aggregates through extensive head domain associations (middle row). Additionally, the short S-2 tail segment was often kinked or coiled and was not well resolved from the fused heads (middle, yellow arrows). In the presence of DUNC-45, the amount of HMM molecules forming heat-induced aggregates was dramatically reduced (bottom row).