Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes

Abstract

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

Keywords: Chaperones; HSP90AA1; HSPA1A; HSPA8; Hsp70; Liposomes; Membranes; Phospholipids.

Fig. 1

Purification and characterization of recombinant HSPA8. HSPA8 (A) and HSPA1 (B) were purified using Ni²⁺-affinity chromatography in one step as described under Methods. All steps for the production and isolation process were visualized by LDS-PAGE and Coomassie Brilliant Blue R-250 staining: lane 1, molecular weight markers: lane 2, bacterial lysate without induction; lane 3, bacterial lysate after IPTG induction; lane 4, supernatant of the bacteria lysate; lane 5, purified recombinant protein. Protein secondary and tertiary structures were assessed by circular dichroism and intrinsic tryptophan fluorescence as described under Methods (C). ATPase activity of both HSPA8 and HSPA1 was measured as described under Methods (D); notice that the activity is elevated for HSPA8 in comparison with HSPA1A. These results all together showed that HSPA8 and HSPA1A used in this study were produced with high purity, presenting the expected structure and function

Fig. 2

HSPA8 and HSPA1 interact preferentially with negatively charged liposomes. A HSPA8 (4 μg) and B HSPA1A (4 μg) were incubated with liposomes made of POPC, POPE, POPS, and CL (400 μg) in 50 mM Tris-HCl buffer (pH 7.5) for 30 min at 25 °C with continuous agitation. The mixture was centrifuged at 100,000×g for 1 h at 4 °C. The pellet was resuspended (300 μL) in 100 mM Na₂CO₃ buffer pH 11.5 and centrifuged at 100,000×g for 1 h at 4 °C in order to eliminate non-specific bound proteins. Hsp70-liposomes were solubilized in sample buffer containing 10 mM β-M, and the proteins were resolved by LDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. The total amount of protein used for the analysis is indicated by all negative signals on the figure legend, whereas the amount of protein incorporated into the liposomes is indicated by the positive signal for each type of phospholipid. C The GelQuant software (http://biochemlabsolutions.com/GelQuantNET.html) was used to estimate the intensity of bands in the gel and the percentage of HSPA8 or HSPA1A incorporated into the liposomes as expressed by a plot for the comparison for insertion of both proteins. D, E Protein secondary and tertiary structures were assessed by circular dichroism and intrinsic tryptophan fluorescence (insets) as described under Methods and did not shown variations in Hsp70s APO or binding into POPS liposomes

Fig. 3

HSPA8 interaction with negatively charged phospholipids is influenced by the protein net charge. A HSPA8 (4 μg) was incubated with POPS or CL liposomes (400 μg) in 50 mM Tris-HCl buffer (pH 7.5), 50 mM Tris-HCl buffer (pH 9.0), or 50 mM acetate buffer (pH = 5.0) for 30 min at 25 °C with continuous agitation. The mixture was centrifuged at 100,000×g for 1 h at 4 °C. The pellet was resuspended (300 μL) in 100 mM Na₂CO₃ buffer pH 11.5 and centrifuged at 100,000×g for 1 h at 4 °C. HSPA8 liposomes were solubilized in sample buffer containing 10 mM βM, and the proteins were resolved by LDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. The GelQuant software (http://biochemlabsolutions.com/GelQuantNET.html) was used to estimate the percentage of HSPA8 incorporated into the liposomes. The results showed an increment of the HSPA8 incorporation in POPS (65 to 95%) and CL liposomes (78 to 87%) at pH 5.0, and a decrease (50%) at pH 9.0. B The total net charge of the HSPA8 at various pHs over the isoelectric point (pH 5.6) was calculated. These data suggest that the HSPA8 charge surface plays an important role in its interaction into liposomes made with negatively charged phospholipids

Fig. 4

HSPA8 interaction with POPS and CL liposomes is driven by entropic changes with a small enthalpy contribution. Thermodynamic parameters and dissociation constants were obtained by ITC as described under Methods using an iTC200 microcalorimeter. Seventeen 2-μL aliquots of POPS or CL liposomes at 3 mM were injected into 203.8 μL of 10–15 μmol L⁻¹ HSPA8, at 25 °C. The pure protein was dialyzed to exhaustion against 50 mM Tris-HCl (pH 7.4) buffer, and the liposomes were prepared in the dialyze buffer to avoid mismatch. The experimental isotherm curves were analyzed to obtain the ΔG_app, ΔH_app, -TΔS_app, and K_D. A HSPA8 interaction with POPS liposomes. B HSPA8 interaction with CL liposomes. The HSPA8 insertion into liposomes was entropically and enthalpically driven, with a higher entropic contribution. C Thermodynamic signatures for HSPA8 interaction with POPS and CL show that the interaction has a very discrete enthalpy contribution, and it is pretty much entropically driven

Fig. 5

Human Hsp70s interaction with liposomes made with negatively charged lipids present a slightly different interaction mechanism. Scatter plot of −TΔS against ΔH for the interaction of HSPA1A, HSPA5, HSPA8, and HSPA9 with POPS (A) and CL (B) liposomes. The double dashed lines indicate the points representing possible combinations of ΔH and −TΔS values giving rise to the two different equilibrium constants indicated (K_D = 10 μM and K_D = 100 μM). All the human Hsp70s, constitutive (HSPA5, HSPA8, and HSPA9) and inducible Hsp70 (HSPA1A), interact with POPS and CL liposomes with similar K_D and consequently ΔG. However, the mechanism for each interaction is slightly different, involving different levels of intramolecular (such as hydrogen-bound) and hydrophobic interactions, water release, and conformational changes

Fig. 6

HSPA8 is inserted into POPS lipid bilayer. HSP8 (4 μg) was incubated with POPS liposomes (400 μg) in 50 mM Tris-HCl buffer (pH 7.5) for 30 min at 25 °C with continuous agitation. The mixture was centrifuged at 100,000×g for 1 h at 4 °C. The pellet was resuspended (300 μL) in 100 mM Na₂CO₃ buffer pH 11.5 and centrifuged again at 100,000×g for 1 h at 4 °C. The pellet was resuspended in 50 mM Tris-HCl buffer pH 7.5 and incubated with proteinase K (5 μg mL⁻¹) for 30 min at 25 °C and centrifuged for 1 h at 100,000×g. The pellet was solubilized in sample buffer containing 10 mM βM, and the proteins were resolved by LDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. Brackets indicate low molecular weight peptides retained within the liposomes after protease digestion

Fig. 7

HSPA8 oligomerizes upon membrane insertion into POPS or CL liposomes. HSP8 (4 μg) were incubated with POPS or CL liposomes (400 μg) in 50 mM Tris-HCl buffer (pH 7.5) for 30 min at 25 °C with continuous agitation. The mixture was centrifuged at 100,000×g for 1 h at 4 °C. The pellet was resuspended (300 μL) in 100 mM Na₂CO₃ buffer pH 11.5 and centrifuged again at 100,000×g for 1 h at 4 °C. The pellet was solubilized in sample buffer containing or not 10 mM βM, and the proteins were resolved by LDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. The brackets indicate the presence of oligomers

Fig. 8

The insertion of HSPA8 into POPS or CL liposomes is decreased by the addition of ATP or ADP. HSP8 (4 μg) was incubated with POPS (A) or CL (B) liposomes (400 μg) in 50 mM Tris-HCl buffer (pH 7.5) for 30 min at 25 °C in the presence or absence of ATP (1mM) or ADP (1 mM). The mixture was centrifuged at 100,000×g for 1 h at 4 °C. The pellet was resuspended (300 μL) in 100 mM Na₂CO₃ buffer pH 11.5 and centrifuged again at 100,000×g for 1 h at 4 °C. The pellet was solubilized in sample buffer containing or not 10 mM βM, and the proteins were resolved by LDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250

Fig. 9

HSPA8 drags HSP90 into POPS and CL liposomes. HSP8 (4 μg) and HSP90AA1 (3 μg) were incubated at 25 °C for 30 min. Then, the mixture was incubated with POPS (A) or CL (B) liposomes (400 μg) in 50 mM Tris-HCl buffer (pH 7.5) for 30 min at 25 °C with continuous agitation. The mixture was centrifuged at 100,000×g for 1 h at 4 °C. The pellet was resuspended (300 μL) in 100 mM Na₂CO₃ buffer pH 11.5 and centrifuged again at 100,000×g for 1 h at 4 °C. The pellet was solubilized in sample buffer containing or not 10 mM βM, and the proteins were resolved by LDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. Notice that HSP90AA1 is only present within liposomes after incubation with HSPA8, suggesting membrane chaperone activity

Fig. 10

Conformational changes of HSPA8 in the presence of nucleotides. A homology model for HSPA8 (1-646 a.a.) in the closed and open states was obtained using the Swiss-Model program (SWISS-MODEL (expasy.org). For closed state, the PDBs templates 3C7N.1.B (HSPA8 covered 1-554 a.a. with 100% of identity) and 2KHO.1.A (HSPA8 covered 1-612 a.a. with 50% of identity) were employed. For open conformation, the templates used were 6ASY.1.A (HSPA8 covered 1-606 a.a. with 66% of identity). The same online tool was also used to generate the HSPA8 N-terminal models (1-401 a.a.). The model in the presence of ATP was generated using the PDB template 6ASY.1.A (HSP8 N-terminal covered 1-401 a.a. with 71% of identity) and for the model in the presence of ADP, the PDB template was used: 3FE1.1.A (HSP8 N-terminal covered 1-401 a.a. with 83% of identity)