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. 2019 May;33(5):6354-6364.
doi: 10.1096/fj.201802182R. Epub 2019 Feb 20.

Maspin binds to cardiolipin in mitochondria and triggers apoptosis

Affiliations

Maspin binds to cardiolipin in mitochondria and triggers apoptosis

Nitin Mahajan et al. FASEB J. 2019 May.

Abstract

A central question in cell biology is how cells respond to stress signals and biochemically regulate apoptosis. One critical pathway involves the change of mitochondrial function and release of cytochrome c to initiate apoptosis. In response to apoptotic stimuli, we found that maspin-a noninhibitory member of the serine protease inhibitor superfamily-translocates from the cytosol to mitochondria and binds to cardiolipin in the inner mitochondrial membrane. Biolayer interferometry assay revealed that recombinant maspin binds cardiolipin with an apparent Kd,of ∼15.8 μM and competes with cytochrome c (apparent Kd of ∼1.31 μM) for binding to cardiolipin-enriched membranes. A hydrophobic, lysine-rich domain in maspin consists of 27 aa, is located at position 268-294, and is responsible for the interaction of this protein with cardiolipin. Depletion of cardiolipin in cells significantly prevents maspin binding to the inner mitochondrial membrane and decreases cytochrome c release and apoptosis. Alteration to maspin's cardiolipin binding domain changes its ability to bind cardiolipin, and tumor cells expressing this mutant have a low frequency of apoptosis. We propose a model of apoptosis in which maspin binds to cardiolipin, displaces cytochrome c from the membrane, and facilitates its release to the cytoplasm.-Mahajan, N., Hoover, B., Rajendram, M., Shi, H. Y., Kawasaki, K., Weibel, D. B., Zhang, M. Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

Keywords: anionic phospholipids; cytochrome; diphosphatidylglycerol; mitochondrial membranes.

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Conflict of interest statement

The authors thank Dr. M. Geiger (Medical University of Vienna, Vienna, Austria) for stimulating discussions and for providing the protein-lipid overlay assay protocol. This work was supported by the U.S. National Institutes of Health (NIH) National Cancer Institute (Grant CA079736 to M.Z.), NIH Office of the Director (Grant 1DP2OD008735-01 to D.B.W.) and the U.S. National Science Foundation (Grant DMR-1121288; University of Wisconsin–Madison). M.R. was supported by the Dr. James Chieh-Hsia Mao Wisconsin Distinguished Graduate Fellowship, B.H. by an NIH Molecular Biophysics Predoctoral Traineeship (T32 GM08293), and H.Y.S by the Northwestern University Zell Foundation. The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Maspin binds to CL. A) A protein-lipid overlay assay demonstrating the interaction between maspin and various lipids [phospholipids (PL), phosphatidylethanolamine (PE), PC, CL, phosphatidic acid (PA), BSA (Mock)]. Wild-type maspin (GST.MpWT) binds specifically to CL and not to the other lipids tested. GST protein (as control) did not bind to the lipids. B) Binding of maspin to CL was determined by retardation of protein mobility in a PAGE–Western blot (WB). T20, Tween 20. Incubation of GST.MpWT with CL reduced the mobility of the protein in contrast to the other lipids. GST was used as a control. C) Dependence of the equilibrium and maximal wavelength shift (Req and Rmax) on protein concentration. Liposomes (50:50 PC:CL by molar concentration) were immobilized on the surface of APS biosensor tips. The interaction between vesicles, cytochrome c (solid line), and maspin (dotted line) was monitored until equilibrium was reached in 25 mM HEPES pH 7.4. Fit to Eq. 1 yielded the apparent association constants (Ka) of 6.9 × 105 M−1 and 6.3 × 104 M−1 for cytochrome c and maspin, respectively. All data points represent means ± sd (n = 3). Representative figures are shown of experiments completed a minimum of 6 times.
Figure 2
Figure 2
Maspin binds CL through a CLBD. A) A schematic representing 6 different putative CLBDs (highlighted with blue, yellow, and red bars) in full-length maspin and truncated mutants containing protein sequences rich in hydrophobic amino acids and Lys residues. B) Comparison of the binding of different constructs of maspin to CL using a protein-lipid overlay assay. Only MpWT bound to CL. C) A schematic depicting a maspin construct in which the 7 Lys residues in the CLBD (region highlighted in red) were swapped with the corresponding sequence from the serpin family member, PAI-1 (pink box). A sequence alignment of maspin (AAA18957.1) and PAI-1 (AAA60009.1) demonstrates the homology of these 2 domains in which 7 Lys residues are present in this region of maspin (red box, aa 268–294) and 2 Lys residues in PAI-1 (pink box, aa 298–324). D) Three-dimensional structure of maspin (Protein Data Bank ID: 1XU8) highlighting the CLBD (depicted in black) and Lys residues (depicted in purple) using Internal Coordinate Mechanics (ICM) Pro v.3.48 software. E) A comparison of binding of GST.MpWT and GST.MpPS with various lipids [phospholipids (PL), phosphatidylethanolamine (PE), PC, CL, phosphatidic acid (PA), BSA (mock)] demonstrates the importance of the CLBD in maspin for binding CL. F) PAGE-Western blots demonstrate that CL causes a gel shift of wild-type maspin (GST.MpWT). Representative figures are shown of experiments completed a minimum of 6 times.
Figure 3
Figure 3
The cellular CL concentration does not affect maspin translocation into mitochondria; however, it affects cytochrome c release and the activation of caspase 3. A) A plot comparing maspin translocated into mitochondria in PGS-S/cPGS1Mp and PGS-SMp cells after STS treatment. The amount of CL does not obviously increase the translocation of maspin into cells. COX IV was used as mitochondrial loading control. B) A plot comparing caspase 3 activity [optical density at 405 nm (OD405)] in PGS-S/cPGS1Mp and PGS-SMp cell lysates in the absence and presence of STS using a colorimetric assay. The data indicate that a 2–3-fold reduction in cellular CL reduces the caspase activity by ∼50%. C) A plot comparing cytochrome c (Cyt C) release in PGS-S/cPGS1Mp and PGS-SMp cells after apoptosis induction indicates that reduction in CL leads to a large decrease in cytochrome c in the cytosol.
Figure 4
Figure 4
Maspin binds CL in the mitochondria and displaces CL-bound cytochrome c (Cyt C). A) A plot comparing the amount of maspin retained in mitochondria isolated from PGS-S/cPGS1 and PGS-S cells; COX IV was used as a mitochondrial loading control. The data indicate that a reduction in cellular CL reduces the concentration of mitochondrial maspin. B) A plot comparing cytochrome c (from the intermitochondrial membrane space) released after ATR treatment of mitochondria isolated from mouse liver. The supernatants were concentrated in a MicroVac and loaded on SDS-PAGE to determine the relative concentration of cytochrome c. ATR treatment led to a large increase in cytochrome c. C) A plot of the amount of cytochrome c released from ATR-treated mitochondria incubated with GST.MpWT compared with GST. The supernatants were concentrated in a MicroVac and loaded on SDS-PAGE to determine the relative concentration of cytochrome c; GST was used as a negative control. D) A plot comparing IMM-bound cytochrome c in mitochondrial pellets after incubating organelles with either GST or GST.MpWT.
Figure 5
Figure 5
Maspin (mas) occludes and displaces cytochrome c (Cyt c) from binding to PC and CL liposomes. A) To measure the competition of cytochrome c and maspin at membranes, we dipped APS-coated BLI biosensors containing adsorbed PC:CL liposomes (50:50 by molar concentration) into wells containing varying ratios of cytochrome c and maspin and a total protein concentration of 5 μM. We measured the competition of protein for the membrane surface for 4200 s. Cytochrome c and maspin (5 μM) are shown in black and dark blue, respectively. Decreasing concentrations of maspin (cytochrome c:maspin molar ratios of 1:1, 2:1, 4:1, and 8:1) are indicated by the color gradient (dark to light blue). Lines indicate the mean wavelength shift values (based on n = 3 measurements), and the shading indicates the the mean ± sd. B) To measure the displacement of cytochrome c by maspin, we equilibrated APS-coated BLI biosensors containing adsorbed PC:CL liposomes (50:50 by molar concentration) into wells containing cytochrome c (5 μM) for 2000 s, then dipped into wells containing 5 μM cytochrome and different concentrations of maspin. The black curve depicts data for cytochrome c (5 μM) dipped into a well containing 5 μM cytochrome c (as a control). Increasing concentrations of maspin are indicated by the color gradient (from light to dark blue). The competition of maspin and cytochrome c was monitored for 7000 s before returning tips to baseline wells to monitor dissociation (unpublished results). Lines indicate the means of 6 experiments, and shading indicates the mean ± sd (A, B).
Figure 6
Figure 6
TM40DPS cells expressing mutant maspin lacking the CLBD translocate to mitochondria and display a low apoptosis efficiency. A) A plot comparing maspin translocated to mitochondria in TM40DMp (MpWT) and TM40DPS cells (containing the corresponding sequence from PAI-1 swapped with the CLBD in maspin) after STS treatment; COX IV was used as a mitochondrial loading control. B) A plot comparing caspase 3 activity [optical density at 405 nm (OD405)] in TM40DMp and TM40DPS cell lysates in the absence and presence of STS using a Colorimetric Assay Kit. C) A plot comparing cytochrome c (Cyt C) released into the cytosol was determined in TM40DMp and TM40DPS cells after apoptosis induction. GAPDH was as used as a cytosolic loading control.
Figure 7
Figure 7
Proposed model for maspin-CL binding in the IMM and release of cytochrome c into the cytoplasm A) Cytochrome c (Cyt C) is bound to CL (depicted in red) in the IMM. Maspin (Mp) is in the cytosol. B) Apoptosis is induced and maspin translocated from the cytosol into mitochondria. C) Maspin competes with cytochrome c for binding to CL in the IMM, and cytochrome c is released from mitochondria and triggers apoptosis.

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