Mitochondria are the power stations of the cell and generate cellular energy in the form of adenosine triphosphate (ATP). ATP is produced by oxidative phosphorylation (OXPHOS) through the mitochondrial electron transport system (ETS) comprising five multisubunit protein complexes, complex I (CI) to complex IV, and complex V, ATP synthase. Mitochondrial dysfunction is associated with many human diseases which can manifest in any organ or tissue, including the nervous system, skeletal and cardiac muscles, kidneys, liver, and endocrine system, and present diverse clinical symptoms (1). There are few effective treatments for mitochondrial disease, as dysfunction of the ETS and OXPHOS for ATP production involves multiple proteins and multiple steps (2). A conventional therapeutic strategy of targeting a single protein is unlikely to succeed in restoring function in complicated mitochondrial diseases. Another significant challenge for treating mitochondrial dysfunction is the efficient delivery of therapeutic molecules to the mitochondria. Elamipretide (SS-31) is a synthetic tetrapeptide that is a member of an emerging new class of therapeutics that selectively target mitochondria to restore mitochondrial bioenergetics. SS-31 is currently under clinical trials for multiple mitochondrial disorders, including aging, mitochondrial genetic disease, ischemia, acute kidney injury, and heart failure (3-5). SS-31 has shown promising results for treatment of various mitochondrial diseases; however, its mechanism of action remains unclear.
Mitochondria are composed of two membranes, an outer mitochondrial membrane (OMM) and an inner mitochondrial membrane (IMM) which forms invaginations, commonly named cristae. The cristae membranes constitute the backbone platform where mitochondrial respiratory complexes are located and OXPHOS takes place. Cardiolipin (CL), an unusual anionic lipid with two phosphate head groups and four acyl chains, is almost exclusively localized on the IMM (6). It has long been known that CL has a critical role in bioenergetics, cristae morphology, and assembly of respiratory components into higher order supercomplexes (7, 8). Bound CL molecules are required for the enzymatic activities and stabilities of both individual protein subunits and protein supercomplexes involved in mitochondrial respiration. For example, CL plays an essential role in the oligomerization of the c-rings and lubrication of its rotation in ATP synthase (CV), which can influence the stability of cristae structure through dimerization (9, 10); CL acts as glue holding respiratory supercomplexes (CIII and CIV) together and steering their assembly and organization (11, 12); and the binding sites of CL identified close to the proton transfer pathway in CIII and CIV suggest a role of CL in proton uptake through the IMM (13-15). In the case of the ADP/ATP translocase, ADT, three bound CL molecules securely anchor the carrier protein in the IMM and affect ADP/ATP transport activity (16). Previous studies have shown that SS-31 peptide can penetrate the OMM and concentrate in the IMM by selectively binding to CL (3-5, 17). The leading hypothesis is that the alternating aromatic-cationic motif of this synthetic tetrapeptide binds CL via dual interactions, i.e., hydrophobic interaction with acyl chain and electrostatic interactions with anionic phosphate head groups. This specific binding has been validated by fluorescence spectroscopy, isothermal titration calorimetry, and NMR analysis (18, 19). Though not experimentally demonstrated, it was suggested that binding of SS-31 to CL helps induce tighter curvatures of cristae to stabilize the IMM structure and optimize the organization of respiratory chain supercomplexes for enhanced electron transfer and ATP production (5, 17). Given the numerous lines of evidence showing SS-31-CL and CL-protein interactions, one could speculate direct interactions may exist between SS-31 and mitochondrial proteins. SS-31-protein interactions could play a direct or synergistic role with CL interactions in mitochondrial structure, cristae morphology, and ATP synthesis. To date, no empirical data exist to demonstrate SS-31 interacts with mitochondrial proteins, and the mechanistic details of how SS-31 modulates and rejuvenates mitochondrial function have remained elusive.
Chemical cross-linking mass spectrometry (XL-MS) has evolved to be a valuable and widely used tool for studying protein structures and interactions (20-23). Recent development of protein interaction reporter (PIR)-based XL-MS technology in our group has further enabled large-scale identification of protein interactions in complex mixtures from living cells (24-26) and isolated functional organelles (27, 28). Previous studies have shown that SS-31, when added to permeabilized cells or isolated mitochondria, can decrease generation of H2 O2 and increase O2 consumption and ATP production (18, 19, 29-31). To investigate the protein interaction landscape of SS-31, we utilized a N-terminal biotinylated form of SS-31 which allows for affinity enrichment of cross-linked SS-31-protein complexes. PIR-based cross-linkers were applied directly to isolated mitochondria incubated with biotinylated SS-31 (bSS-31) to secure a snapshot of protein interactions in their functional state. Subsequent mass spectrometry analysis uncovered the identities of SS-31 protein interactors with topological information. Here, we present a report of the SS-31 protein interaction network, comprising 12 mitochondrial proteins belonging to nine enzymatic complexes. Interestingly, all 12 proteins were previously reported to bind with CL on the IMM and contribute directly and/or indirectly to mitochondrial respiration. These data provide evidence of direct interactions between SS-31 and CL-associated proteins. Thus, in addition to effects on IMM structure, the interaction between SS-31 and CL may serve to localize SS-31 to the IMM and facilitate its interactions with CL-binding proteins. These SS-31-protein interactions provide insight into the functional role of SS-31 within mitochondria. Furthermore, our research establishes a general approach to investigate the structural details of protein interactions with therapeutic molecules in situ.
Results and Discussion
bSS-31 Interactome.
SS-31 has been shown to selectively interact with CL, localizing and concentrating the peptide in the IMM (18). To identify SS-31 protein interaction partners, we utilized an affinity-tagged version containing a biotin group on the peptide N terminus (bSS-31) (SI Appendix, Fig. S1A) and carried out the experimental workflow illustrated in Fig. 1. SS-31 has been demonstrated to have no effect on mitochondrial function in young healthy mitochondria, while increasing mitochondrial ATP production and reducing H2 O2 production and redox stress in aged mouse muscle (30, 32). Importantly, bSS-31 also increased mitochondrial respiration and reduced H2 O2 production in mitochondrial isolated from aged mice (see Figs. 3 and 4), while not affecting either parameter in mitochondria from young animals. Both SS-31 and bSS-31 reduced superoxide production in aged cardiomyocytes (SI Appendix, Fig. S3B), indicating no discernable difference in the antioxidant activity of SS-31 due to addition to the biotin tag. Isolated mitochondria from aged mouse hearts were incubated with 10 μM bSS-31 for an hour prior to chemical cross-linking with a PIR cross-linker (DP-amide) (Fig. 1 and SI Appendix, Fig. S1B). After cross-linking, mitochondrial proteins were extracted with urea and subjected to a standard tryptic digestion protocol. Peptides cross-linked to bSS-31 were enriched using avidin affinity chromatography and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis employing two different acquisition methods, ReACT (33) and Mango (34) developed in our laboratory for the identification of PIR cross-linked peptide pairs. In total we were able to confidently identify 17 nonredundant cross-linked peptide pairs between bSS-31 and 16 Lys residues on 12 different proteins as shown in Fig. 2 and Dataset S1. The bSS-31 cross-linked peptide identifications were repeatedly observed across biological replicate samples originating from four mice. The cross-linked lysine residues are indicated in Fig. 2. Interestingly the bSS-31 interacting proteins can be grouped into two broad classes, those involved in ATP production and those utilizing 2-oxoglutarate (Fig. 2). Furthermore, each of the 12 proteins is known to directly interact with CL which is important in maintaining the structures and functions for these proteins (Fig. 2).
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