Protection of Membrane Contact Protein by the Methionine Sulfoxide Reductases

Abstract

In this News and Views, I discuss our recent publication that established how steroidogenic acute regulatory-related lipid transfer domain-3 (STARD3), a membrane contact protein situated at lysosomal membranes, plays a role in the detoxification of cholesterol hydroperoxide. STARD3's methionine residues can be oxidized to methionine sulfoxide by cholesterol hydroperoxide, after which methionine sulfoxide reductases reduce the methionine sulfoxide residues back to methionine. The reaction also results in the reduction of the cholesterol hydroperoxide to an alcohol. The cyclic oxidation and reduction of methionine residues in STARD3 at membrane contact sites creates a catalytically efficient mechanism for detoxification of cholesterol hydroperoxide during cholesterol transport, thus protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

A membrane contact site (MCS) is created when two or more organelles come together in close proximity within ∼10 to 30 nm and act as communication hubs, facilitating Ca²⁺ signaling and transferring small molecules such as ions, lipids, and signaling molecules from one organelle to another (Levine, 2004). MCSs have been implicated in various cellular functions, facilitating communication, exchange of materials, and coordination between different organelles in cells (Prinz, 2014; Prinz et al., 2020).

Steroidogenic acute regulatory-related lipid transfer domain-3 (STARD3), a cholesterol-specific protein, plays a crucial role in MCS formation by interacting with multiple organelles. It has distinct structural domains, namely the MENTAL domain (MLN64 N-terminal domain) anchoring the protein in endosomal membranes, the StAR-related lipid-transfer (START) domain binding cholesterol in the cytosol at a 1:1 ratio, and the FFAT motif interacting with vesicle-associated membrane protein-associated protein in the endoplasmic reticulum (ER). These enable STARD3 to form an MCS between the ER and late endosome/lysosome. One of the significant functions of STARD3-mediated MCS formation is the efficient transport of cholesterol between the ER and the lysosome (Alpy et al., 2013; Wilhelm et al., 2017). This process is particularly relevant in Niemann-Pick type C protein 1 (NPC1)-deficient cells, where a reduction in STARD3 levels drastically reduces the contact between lysosomes and mitochondria. This suggests a critical role for STARD3 in forming these lyso-mitochondria MCSs (Höglinger et al., 2019). The findings in NPC1-deficient cells raise the possibility that STARD3 might also play a crucial role in normal cells regarding the formation of MCSs between lysosomes and mitochondria. However, further research is necessary to test this speculation and elucidate the extent of STARD3's involvement in MCS formation in nondisease states.

Cholesterol is essential for maintaining membrane integrity and acts as a precursor of various cell signaling molecules. However, under oxidative conditions, cholesterol readily undergoes oxidation, forming cholesterol hydroperoxides (ChOOHs), which are reactive compounds that can cause damage to cellular components and organelles. In addition to cholesterol being transported from the ER to the endosome by STARD3, cholesterol hydroperoxide is presumably also transferred through ER-endosome MCS (Figure 1). ChOOHs have deleterious effects and can translocate to other membranes and cells, thereby extending their damaging effects (Girotti and Korytowski, 2021). For instance, steroidogenic acute regulatory (StAR) proteins in steroidogenic cells are responsible for the delivery of cholesterol from the outer to inner mitochondrial membrane for the synthesis of steroid hormones (Miller, 2007a, b). Under oxidative stress, StAR proteins in MA-10 Leydig cells loaded mitochondria with ChOOHs, causing a loss of mitochondrial membrane potential (Korytowski et al., 2013). Despite these findings, the specific details regarding how ChOOHs are transported through MCS between organelles, such as from the ER to the mitochondria, and whether STARD3 or other proteins play a direct role in this process remain unclear. The pathway shown in Figure 1 proposes such transport but lacks explicit elucidation of the molecular mechanisms or proteins involved in ChOOHs trafficking through MCSs. Elucidating the dynamics of ChOOHs movement between organelles and identifying the specific proteins or processes involved in this transport could provide important insights into the role of lipid peroxidation in cellular functions and diseases linked to oxidative stress.

Figure 1.

Cholesterol transport by STARD3 from ER to late endosome/lysosome, and mitochondria. STARD3 is shown to transfer Ch/ChOOH (depicted by a solid arrow) from the ER to a LE/lysosome, indicating the protein’s role in shuttling cholesterol and its oxidized form within the cell, and the potential subsequent delivery of Ch/ChOOH to mitochondria through interorganelle contacts (as denoted by the gray dashed arrow). However, the precise mechanism by which STARD3 mediates the transfer of cholesterol to mitochondria is not understood. MSRs play a role in repairing oxidized STARD3, potentially restoring its function. This repair mechanism could play a crucial role in maintaining STARD3's ability to carry out cholesterol transport and regulate ChOOH, ensuring the proper functioning of cellular lipid homeostasis and defense against oxidative stress.

Lipid hydroperoxides (LOOHs) including ChOOHs, generated through the oxidation of polyunsaturated fatty acids, are central to the process of ferroptosis. Ferroptosis is a form of programmed cell death that is characterized by iron-dependent accumulation of lipid peroxides, which can lead to cell membrane damage and subsequent cell death (Stockwell et al., 2017). Studies have implicated specific regulators of ferroptosis, such as glutathione peroxidase 4 (GPX4), in protecting cells against cholesterol hydroperoxide-induced cell death. Until recently, GPX4 was considered the sole enzyme protecting cells from ChOOH-induced cell death by reducing LOOHs. The specific pathways involved in the detoxification of cholesterol peroxides have not been well explored.

Oxidative stress and ChOOH accumulation can also disrupt the proper function of MCSs. Dysfunction in MCSs may cause a multitude of disrupted functions, including metabolic disorders, impaired mitochondrial function, disturbances in calcium ion homeostasis, and neuronal dysfunction and degeneration (Guillén-Samander and De Camilli, 2023). However, there is a lack of understanding of how MCS deals with deleterious ChOOHs and is linked with antioxidant enzymes for detoxification. Understanding how contact proteins such as STARD3 and MCSs respond to or are affected by oxidative stress would be important for many cellular processes, particularly those related to lipid trafficking and organelle communication. That understanding may identify new therapeutic targets for diseases in which MCS dysfunction contributes to the pathology of the disease.

In our recent study, we investigated how STARD3 and methionine sulfoxide reductase (MSR) cooperate to reduce ChOOHs to their alcohols (Lim et al., 2023). We previously reported that STARD3 is an in vivo binding partner of methionine sulfoxide reductase A (MSRA). When STARD3's methionine residues were oxidized to methionine sulfoxide by hypochlorite, MSRA reduced them back to methionine (Lim et al., 2018). We then hypothesized that ChOOHs could bind to STARD3 that they would also oxidize the protein's methionine residues, and that MSR could repair the protein by reducing the sulfoxide back to methionine. In its cytosolic START domain, STARD3 contains two methionine residues, Met307 and Met427. Met307 is located at the end of the binding pocket for cholesterol, and it is necessary for cholesterol to bind to the protein (Tsujishita and Hurley, 2000). The sulfur of Met307 in STARD3 is only 5.5 from the C6 carbon of cholesterol. This suggests that a hydroperoxide at C5, C6, or C7 could oxidize Met307 of STARD3 and deleteriously affect its ability to transport cholesterol. Met427 is in the C-terminal helix α4, which forms part of the tunnel roof and is solvent-exposed.

To test the hypothesis, we first synthesized and purified ChOOHs. There are four isomers of ChOOHs produced: 7α-hydroperoxy-3β-hydroxycholest-5-ene (7α-OOH), 5α-hydroperoxy-3β-hydroxycholest-6-ene (5α-OOH), 6α-hydroperoxy-3β-hydroxycholest-4-ene (6α-OOH), and 6β-hydroperoxy-3β-hydroxycholest-4-ene (6β-OOH). Using liquid chromatography-mass spectrometry, we found that both Met307 and Met427 are susceptible to oxidation by 6α-OOH and 7α-OOH, while 5α-OOH and 6β-OOH were unreactive. Next, we investigated whether MSRs were capable of reducing the two methionine sulfoxides found on STARD3 back to Met. There are four MSRs in mammals, MSRA, and three methionine sulfoxide reductase Bs (MSRBs). The MSRA is stereospecific for the S-epimer, while the MSRBs are stereospecific for the R-epimer. We showed that not just MSRA, but all three MSRBs are bound to STARD3. These enzymes are distributed in various cellular compartments so they would need to translocate to the endosomal membrane to bind to STARD3. We showed by confocal microscopy that all four green fluorescent protein-tagged MSRs are enriched at the surface of the late endosome/lysosome in HeLa cells overexpressing STARD3. When oxidized STARD3 was incubated with MSRA alone or MSRB alone, ∼50% of methionine sulfoxide was reduced, consistent with their distinct specificity, respectively, for the S- or R-epimers of methionine sulfoxide. When incubated with both MSRA and MSRB, oxidized STARD3 was completely reduced. The repaired STARD3 molecule should again be able to bind cholesterol or its hydroperoxide at MCS, although we did not test for restored binding. Thus, the STARD3/MSR system provides a catalytically efficient mechanism for detoxifying ChOOHs (Figure 2).

Figure 2.

STARD3 and MSR constitute a catalytically efficient system for detoxification of cholesterol hydroperoxide at MCS. STARD3 acts as a shuttle, binding to ChOOH and transferring it between organelles while being subject to oxidation, whereas MSRs serve to repair and regenerate STARD3, ensuring its continued ability to neutralize ChOOH.

In summary, we have found that all four mammalian MSRs bind to STARD3 at the membrane of the late endosome/lysosome. Upon binding to a cholesterol hydroperoxide, STARD3's methionine residues can be oxidized to sulfoxide, a reaction that also reduces the cholesterol hydroperoxide into a nontoxic alcohol. The MSRs then play a pivotal role in reducing the methionine sulfoxide residues in StARD3 back to methionine, thereby regenerating the protein and completing the cycle of oxidation and reduction. The result of the cyclic process of oxidation and reduction of methionine residues in StARD3 is to help in the scavenging and detoxification of toxic ChOOHs. The StARD3–MSR collaboration in detoxifying ChOOHs represents a significant breakthrough in understanding cellular defense mechanisms against oxidative stress induced by LOOHs and suggests that enhancing activity or expression of this system could be a potential therapeutic strategy for diseases associated with oxidative stress and ChOOHs accumulation.