Mitochondrial MsrB2 serves as a switch and transducer for mitophagy
2019; Springer Nature; Volume: 11; Issue: 8 Linguagem: Inglês
10.15252/emmm.201910409
ISSN1757-4684
AutoresSeung Hee Lee, Suho Lee, Jing Du, Kanika Jain, Min Ding, Anis John Kadado, Gourg Atteya, Zainab Jaji, Tarun Tyagi, Won‐Ho Kim, Raimund I. Herzog, Amar Patel, Costin Ionescu, Kathleen A. Martin, John Hwa,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle8 July 2019Open Access Source DataTransparent process Mitochondrial MsrB2 serves as a switch and transducer for mitophagy Seung Hee Lee Corresponding Author Seung Hee Lee [email protected] orcid.org/0000-0003-3642-1416 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Division of Cardiovascular Diseases, Center for Biomedical Sciences, National Institute of Health, Cheongju, Chungbuk, Korea Search for more papers by this author Suho Lee Suho Lee Departments of Neurology and Neurobiology, Cellular Neuroscience, Neurodegeneration and Repair Program, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jing Du Jing Du Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Kanika Jain Kanika Jain Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Min Ding Min Ding Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Anis J Kadado Anis J Kadado Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Gourg Atteya Gourg Atteya Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Zainab Jaji Zainab Jaji Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Tarun Tyagi Tarun Tyagi Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Won-ho Kim Won-ho Kim Division of Cardiovascular Diseases, Center for Biomedical Sciences, National Institute of Health, Cheongju, Chungbuk, Korea Search for more papers by this author Raimund I Herzog Raimund I Herzog Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Amar Patel Amar Patel Division of Movement Disorders, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Costin N Ionescu Costin N Ionescu Yale Cardiovascular Medicine, Department of Internal Medicine, Yale-New Haven Hospital, New Haven, CT, USA Search for more papers by this author Kathleen A Martin Kathleen A Martin Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author John Hwa Corresponding Author John Hwa [email protected] orcid.org/0000-0001-7366-2628 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Seung Hee Lee Corresponding Author Seung Hee Lee [email protected] orcid.org/0000-0003-3642-1416 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Division of Cardiovascular Diseases, Center for Biomedical Sciences, National Institute of Health, Cheongju, Chungbuk, Korea Search for more papers by this author Suho Lee Suho Lee Departments of Neurology and Neurobiology, Cellular Neuroscience, Neurodegeneration and Repair Program, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jing Du Jing Du Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Kanika Jain Kanika Jain Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Min Ding Min Ding Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Anis J Kadado Anis J Kadado Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Gourg Atteya Gourg Atteya Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Zainab Jaji Zainab Jaji Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Tarun Tyagi Tarun Tyagi Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Won-ho Kim Won-ho Kim Division of Cardiovascular Diseases, Center for Biomedical Sciences, National Institute of Health, Cheongju, Chungbuk, Korea Search for more papers by this author Raimund I Herzog Raimund I Herzog Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Amar Patel Amar Patel Division of Movement Disorders, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Costin N Ionescu Costin N Ionescu Yale Cardiovascular Medicine, Department of Internal Medicine, Yale-New Haven Hospital, New Haven, CT, USA Search for more papers by this author Kathleen A Martin Kathleen A Martin Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author John Hwa Corresponding Author John Hwa [email protected] orcid.org/0000-0001-7366-2628 Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Author Information Seung Hee Lee *,1,2, Suho Lee3, Jing Du1, Kanika Jain1, Min Ding1, Anis J Kadado1, Gourg Atteya1, Zainab Jaji1, Tarun Tyagi1, Won-ho Kim2, Raimund I Herzog4, Amar Patel5, Costin N Ionescu6, Kathleen A Martin1 and John Hwa *,1 1Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA 2Division of Cardiovascular Diseases, Center for Biomedical Sciences, National Institute of Health, Cheongju, Chungbuk, Korea 3Departments of Neurology and Neurobiology, Cellular Neuroscience, Neurodegeneration and Repair Program, Yale University School of Medicine, New Haven, CT, USA 4Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA 5Division of Movement Disorders, Departments of Neurology and Neurobiology, Yale University School of Medicine, New Haven, CT, USA 6Yale Cardiovascular Medicine, Department of Internal Medicine, Yale-New Haven Hospital, New Haven, CT, USA *Corresponding author. Tel: +82 43 719 8664; E-mail: [email protected] *Corresponding author. Tel: +1 203 737 5583; E-mail: [email protected] EMBO Mol Med (2019)11:e10409https://doi.org/10.15252/emmm.201910409 See also: A Kaur & EE Gardiner (August 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitophagy can selectively remove damaged toxic mitochondria, protecting a cell from apoptosis. The molecular spatial–temporal mechanisms governing autophagosomal selection of reactive oxygen species (ROS)-damaged mitochondria, particularly in a platelet (no genomic DNA for transcriptional regulation), remain unclear. We now report that the mitochondrial matrix protein MsrB2 plays an important role in switching on mitophagy by reducing Parkin methionine oxidation (MetO), and transducing mitophagy through ubiquitination by Parkin and interacting with LC3. This biochemical signaling only occurs at damaged mitochondria where MsrB2 is released from the mitochondrial matrix. MsrB2 platelet-specific knockout and in vivo peptide inhibition of the MsrB2/LC3 interaction lead to reduced mitophagy and increased platelet apoptosis. Pathophysiological importance is highlighted in human subjects, where increased MsrB2 expression in diabetes mellitus leads to increased platelet mitophagy, and in platelets from Parkinson's disease patients, where reduced MsrB2 expression is associated with reduced mitophagy. Moreover, Parkin mutations at Met192 are associated with Parkinson's disease, highlighting the structural sensitivity at the Met192 position. Release of the enzyme MsrB2 from damaged mitochondria, initiating autophagosome formation, represents a novel regulatory mechanism for oxidative stress-induced mitophagy. Synopsis Mitophagy can be selectively switched on at sites of oxidatively damaged mitochondria: MsrB2 mediates these actions by switching on Parkin and transducing activation through LC3. This mechanism in found in diabetic platelets and may be important for Parkinson's disease. MsrB2 removes reduces oxidized methionine back to its native state and is induced in diabetic platelets. Parkin Met192 is oxidized in a high oxidative stress environment leading to Parkin dysfunction, aggregation and prevention of mitophagy. MsrB2 released from damaged mitochondria reduces MetO192 restoring Parkin's function (switch), allowing ubiquitination of MsrB2 and interaction with LC3 (transducer). The process of mitophagy is restored, protecting against apoptosis. The association of mutation of Met192 with Parkinson's disease led to studies that demonstrate a reduction in MsrB2 in patients with Parkinson's disease. Introduction Currently, over 19.7 million adults in the USA have diagnosed DM and an estimated 8.2 million have undiagnosed DM (Benjamin et al, 2017). Diabetes mellitus (DM) is a progressive and chronic metabolic disorder characterized by hyperglycemia arising from impaired insulin levels, insulin sensitivity, and/or insulin action, leading to increased oxidative stress. Sixty-five percent of patients with DM will die from thrombotic events including heart attacks and strokes (Ferreiro & Angiolillo, 2011) where oxidative stress and platelets play a major role (Tang et al, 2011, 2014). Platelets are short-lived (7–10 days), small (2–4 μm), circulating anucleate cells, containing many critical factors required for regulation of thrombus formation, vascular homeostasis, and immune response (Lindemann et al, 2001; Leytin, 2012). Despite having no genomic DNA and thus limited transcriptional capabilities, platelets are capable of many fundamental cellular functions including de novo protein synthesis (Weyrich et al, 1998; Lindemann et al, 2001), programmed cell death (Mason et al, 2007), and autophagy (Feng et al, 2014; Ouseph et al, 2015; Lee et al, 2016). Platelets are prepackaged with the relevant mRNAs to allow for these well-orchestrated protective autophagy processes to maintain normal cellular function (basal autophagy) and to protect (induced autophagy) from severe oxidative stressors, as observed with diabetes mellitus or high-fat diet (Lee et al, 2016). Autophagy and mitophagy play many emerging diverse critical roles in stemness and senescence (Palikaras et al, 2015; Garcia-Prat et al, 2016), progression to cell death in mitosis (Domenech et al, 2015), and resistance against infection (Manzanillo et al, 2013) through Parkin-dependent and Parkin-independent processes. Parkin, a ubiquitin E3 ligase, adds ubiquitin to many substrates leading to interactions with LC3, a key component of autophagosomes (Gegg et al, 2010; Geisler et al, 2010; Kane & Youle, 2011; Lee et al, 2016). Mitophagy is induced in human diabetic platelets through a PINK1- and Parkin-dependent process (Lee et al, 2016). The process of Parkin-dependent mitophagy is initiated by PINK1 accumulation in the OMM in response to mitochondrial depolarization and damage. PINK1 phosphorylates ubiquitin to activate the E3 ligase function of Parkin (Koyano et al, 2014) as well as the outer mitochondrial membrane protein MFN2 to serve as a Parkin receptor (Chen & Dorn, 2013). Once recruited, Parkin ubiquitinates multiple outer mitochondrial membrane proteins (e.g., VDAC1, HDAC6, and mitofusin) leading to interaction with LC3 and initiation of mitophagy (Shaid et al, 2013). In the high-oxidative stress DM environment, the molecular mechanism for selective removal of damaged mitochondria sparing adjacent intact mitochondria remains unclear. We now report that the mitochondrial matrix protein methionine sulfoxide reductase B2 (MsrB2) is a Parkin substrate necessary for mitophagy induction. MsrB2 released from ruptured mitochondria reduces oxidized Parkin. In the absence of MsrB2, oxidized Parkin is inactive (aggregates). Whereas PINK1 accumulation identifies mitochondria undergoing oxidative stress, MsrB2–Parkin protein–protein interaction serves as a switch mechanism, allowing mitophagy to proceed only in mitochondria that are severely damaged or ruptured. This mechanism appears to occur in other nucleated cells. Results Two mitochondria are residing side by side in the high-oxidative stress environment associated with diabetes mellitus: One is morphologically intact, while the other is severely damaged with loss of crista structure, swelling, and rupture (Fig 1A). A double membrane envelope (highlighted in red) appears to be forming at the site of loss of structural integrity. A key intriguing question is the molecular spatio-temporal mechanism that allows selection of the damaged mitochondria for autophagy, sparing the adjacent intact mitochondria. Such a mechanism may be critical for protection from apoptosis and improved cell survival. Figure 1. Identification of MsrB2 as LC3 interaction protein in human platelets A. Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of crista morphology) next to the intact mitochondria. c. Black dashed line indicates mitochondrial membrane, and red dashed line indicates phagophore formation (M, mitochondria). * means loss of structural integrity of mitochondria membrane. B. Immunoprecipitation (Meng et al, 2011) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II Western blot analysis. C. Confocal microscopy was used to corroborate the Western blot analysis using double staining for LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of CoxIV, LC3, and MsrB2 as determined by the Volocity software (PerkinElmer, USA). Quantification is presented for the intensity of MsrB2 in HC vs DM and colocalization of CoxIV, LC3, and MsrB2 (MsrB2 intensity, **P = 0.0009; colocalization CoxIV and LC3, **P = 0.0014 vs. HC; n = 3). The nonparametric t-test was performed for comparisons of two groups. Analysis was performed with Prism software (GraphPad Software, Inc., La Jolla, CA). A difference of P < 0.05 was considered significant (mean ± SD). D. LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15-nm dots indicate immunogold-labeled LC3 clusters, and 5-nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b–d) are presented. Download figure Download PowerPoint MsrB2 interacts with LC3 in DM platelets We initially set out to determine unique requirements for the intriguing nucleus- and transcription-independent mitophagy process in DM platelets. As LC3 is a central component of autophagosome formation, we sought LC3-interacting proteins by inducing platelet mitophagy with CCCP, and immunoprecipitating LC3, followed by mass spectrometry (Appendix Fig S1A). We identified an unlikely interaction with methionine sulfoxide reductase B2 (MsrB2). MsrB2 reduces methionine sulfoxidation under conditions of severe oxidative stress (Fischer et al, 2012). However, MsrB2 resides in the mitochondrial matrix (Pascual et al, 2010; Ugarte et al, 2010; Fischer et al, 2012), and LC3 is a cytoplasmic membrane protein, suggesting our result may be an artifact of intense membrane solubilization, a common problem associated with immunoprecipitation. However, we noted multiple distinct LC3-interacting motifs (LIFs; Valdor & Macian, 2012) in both human MsrB2 (3 LIFs) and mouse MsrB2 (2 LIFs; Appendix Fig S1B). Moreover, MsrB2 was significantly induced in DM platelets, in contrast to MsrA and MsrB1 and MsrB3 (Appendix Fig S1C). Further exploration was warranted. Given the potential pathophysiological relevance, we elected to determine native interactions in human DM patient platelets (non-DM served as the control), and rather than overexpressing proteins in cell culture systems, we performed the converse of the initial experiments, immunoprecipitating MsrB2. We detected an LC3 band (particularly LC3II, lower band) in diabetic human platelets (Fig 1B). To further support an MsrB2/LC3 interaction, we used high-resolution confocal microscopy. Individual platelets in situ demonstrated significant MsrB2 and LC3 colocalization only in DM platelets (Fig 1C). Moreover, immunoelectron microscopy demonstrated colocalization of small (5 nm; MsrB2) and large particles (15 nm; LC3) at mitochondria in DM platelets, with no significant colocalization in WT control (Fig 1D). Taken together, the selective increase in MsrB2 in DM platelets, the dual co-IP of LC3 and MsrB2, the LIFs on MsrB2, and the colocalization in platelets using confocal microscopy and immunoelectron microscopy all supported an MsrB2/LC3 interaction. MsrB2 may play a role in the mitophagy process. MsrB2 knockout leads to reduced mitophagy and increased platelet apoptosis The interaction between MsrB2 and LC3 supports a role for MsrB2 in the regulation of mitophagy. MsrB2 knockdown (shMsrB2) significantly reduced mitophagy (LC3II, lower band) and mitophagy induced by H2O2 (Fig 2A). We additionally used a high-glucose (25 mM) stress, previously demonstrated to oxidatively stress platelets and induce a protective mitophagy response (Tang et al, 2014). Platelet mitophagy is recognized to protect against platelet apoptosis (Lee et al, 2016). Thus, with loss of mitophagy, we would expect a platelet apoptosis phenotype. Knockdown of MsrB2 leads to reduced mitophagy (LC3II, lower band) and significant increases in pro-apoptotic pp53(S15) (Fig 2B). Given our ex vivo immunoprecipitation results demonstrating an interaction between MsrB2 and LC3, and our in vitro knockdown results supporting a role for MsrB2 in mitophagy and thus preventing apoptosis, we then assessed for platelet apoptosis, in vivo. A global MsrB2 knockout mouse supported increased platelet apoptosis. An initial apoptosis array was followed by evidence of increased cytochrome c release (Appendix Fig S2A and B). Although these results support a role for MsrB2 in mitophagy, endothelial dysfunction (arising from MsrB2 knockout) may also lead to platelet apoptosis, and thus, we needed a platelet-selective knockout. Platelet-selective MsrB2 knockout (PF4 Cre MsrB2−/− compared to MsrB2 fl/fl, without the PF4 Cre) demonstrated increased reactive oxygen species (ROS) (Fig 3A) and significant loss of mitochondrial membrane potential (TMRE; Fig 3B and C). Increased platelet apoptosis was observed consistent with the reduced protective platelet mitophagy process (Fig 3D–F). However, as MsrB2 likely reduces MetO from many important mitochondrial proteins, to further demonstrate that the specific MsrB2 interaction with LC3 is important for mitophagy in vivo, we designed cell-penetrating peptides (based upon MsrB2 amino acid sequence; Appendix Fig S3A) to selectively disrupt the MsrB2/LC3-interacting motif (LIF) (Foroud et al, 2003). We included a Tat-control peptide (CP) (Appendix Fig S3A) as a negative control. Initial treatment of MEG-01 cells with a dose response and induction of stress with high glucose demonstrated reduced mitophagy and increased platelet apoptosis (pp53(S15) and active caspase-3) as compared to control peptide (Appendix Fig S3B). After peptide injection (LP or CP) into HFD mice (intraperitoneally for five consecutive days to induce mild/moderate oxidative stress), as anticipated Parkin, LC3I/II, and MsrB2 were all induced secondary to the increased oxidative stress from the HFD (Fig 3G and H). However, the mice treated with the inhibiting peptide (Foroud et al, 2003) (compared to HFD mice treated with control peptide) demonstrated significantly increased platelet apoptosis as assessed by active caspase-3 (Fig 3G and H). Taken together, both the cell culture and in vivo experiments support that MsrB2 interacts with LC3 and may be involved in removing mitochondria in preventing platelet apoptosis. Given the in vivo phenotype, we proceeded to establish the molecular mechanism for this intriguing interaction between a mitochondrial matrix protein (MsrB2) and a cytoplasmic protein (LC3). Figure 2. MsrB2 is needed for mitophagy A. Western blot analysis of MsrB2 and LC3I/II in MEG-01 cells after shMsrB2 transfection (72 h). Cells were then treated with H2O2 (1 mM for 1 h) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control (shMsrB2 vs. shCon, *P = 0.024; shMsrB2/H2O2 vs. shCon/H2O2, *P = 0.0295; shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC, **P = 0.0100; n = 3, mean ± SD). B. Western blot analysis of MsrB2, pp53(S15), and LC3I/II in MEG-01 cells after 25 mM HG treatment for 24 h. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control (MsrB2: shCon/HG vs. shCon, **P = 0.0017; shMsrB2/HG vs. shCon/HG, *P = 0.0189; pp53: shMsrB2/HG vs. shCon/HG, *P = 0.0317; LC3II: shCon/HG vs. shCon, *P = 0.0215; shMsrB2/HG vs. shCon/HG, *P = 0.0158; n = 3, mean ± SD). Data information: The nonparametric t-test was performed for comparisons of two groups. Analysis was performed with Prism software (GraphPad Software, Inc., La Jolla, CA). A difference of P < 0.05 was considered significant. Source data are available online for this figure. Source Data for Figure 2 [emmm201910409-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint Figure 3. Platelet-specific deletion of MsrB2 leads to increased oxidative stress and intraplatelet apoptosis A. Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA (ROS, *P = 0.0139; MsrB2 fl/fl n = 5, MsrB2−/− n = 6). B, C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockout and age-matched floxed mice. Results are expressed as percentage of apoptotic cells (TMRE, *P = 0.0118; MsrB2 fl/fl n = 5, MsrB2−/− n = 5). D. Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41-stained platelets from the KO mice using fluorescent-labeled antibodies against the respective proteins of interest. Magnification: 100×. The rightmost panel represents zoom-in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3. E. Graph indicates platelets positive for active caspase-3 signal. The y-axis indicates percentage of caspase-3-positive cells from total cells calculated from n = 10 independent images per sample (active caspase-3 signal, **P = 0.0003). F. Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n = 6 samples per group. Values expressed as mean ± SEM (caspase-3 activity, *P = 0.0157) G, H. Western blot analysis of Parkin, LC3I/II, MsrB2, and active caspase-3 in platelets after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al, 2003). Quantification analysis of individual band intensity (Parkin, *P = 0.02964; LC3II, *P = 0.03200; MsrB2, **P = 0.0059; active caspase-3, *P = 0.0368 vs. chow or HFD group; each group n = 3). Actin served as the loading control. Data information: The nonparametric t-test was performed for comparisons of two groups. Analysis was performed with Prism software (GraphPad Software, Inc., La Jolla, CA). A difference of P < 0.05 was considered significant. Mean ± SD (A, C, E and H). Download figure Download PowerPoint MsrB2 also interacts with Parkin and prevents aggregation With upregulation of MsrB2 in DM platelets and interaction with LC3 (a key player in DM platelet mitophagy), and mitophagy being a Parkin-dependent process in diabetic platelets (Lee et al, 2016), we then sought to determine whether MsrB2 could also interact with Parkin. Immunoprecipitation of MsrB2 pulled down Parkin on Western blot analysis (Fig 4A). Conversely, immunoprecipitation of Parkin demonstrated a pulldown of MsrB2, particularly in DM platelets (Fig 4B). Confocal microscopy labeling Parkin, LC3, and MsrB2 on individual platelets demonstrated increased colocalization of all three components in DM versus control platelets (Fig 4C and D). This interaction between MsrB2 and key components of mitophagy strongly supports a complex functional relationship between MsrB2 and Parkin-dependent mitophagy. MetO on proteins serves as the substrate for methionine sulfoxide reductases (Msr; Ugarte et al, 2010; Fischer et al, 2012; Gu et al, 2015). Using a MetO antibody (in nonreducing conditions), oligomerized (high molecular weight) Parkin appears to have a MetO and thus may serve as a substrate for MsrB2 (Fig 4E). We then developed an in vitro assay using recombinant MsrB2 and assessing for the effect on Parkin aggregation (induced by H2O2) (Fig 4F). The last two lanes demonstrate that with little MsrB2 expression (red arrow), Parkin oligomerization is apparent. In contrast, substantial MsrB2 expression (blue arrow) prevents Parkin aggregation (Fig 4F). Taken together, the data suggest that in DM platelets, physical interaction can occur between the mitochondrial matrix protein MsrB2 and the outer mitochondrial membrane Parkin. Parkin is MetO-modified and is associated with Parkin aggregation. This aggregation may be prevented by MsrB2 overexpression. Figure 4. MsrB2 interacts with key mitophagy proteins, Parkin and LC3 A. Immunoprecipitation (Meng et al, 2011) of MsrB2 in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and modified Parkin. B. Immunoprecipitation (Meng et al, 2011) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2. C. Confocal microscopy was used to corroborate the Western blot analysis using triple staining for Parkin, LC3, and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3, and MsrB2 as determined by the Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3, and MsrB2 signal. D. Signal intensity of each group was converted to fold change and compared with the HC values (colocalization of Parkin, LC3, and MsrB2, **P = 0.0006). The nonparametric t-test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc., La Jolla, CA). A difference of P < 0.05 was considered significant (mean ± SD, n = 3). E. Immunoprecipitation (Meng et al, 2011) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer. F. In vitro assay. Parkin oxidation was induced with 1 mM H2O2 then incubated with MsrB2 protein at 37°C for 2 h. Western blot analysis of Parkin and MsrB2 was performed (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). Download figure Download PowerPoint Confirmation of MetO on Parkin Reactive oxygen species is significantly increased in DM platelets (Tang et al, 2014; Lee et al, 2016; Fig 5A). Protein modifications including 3-nitrotyrosine, 4-hydroxynonenal, carbonyl derivatives, and polyubiquitination are hallmarks of oxidative stress (Silva et al, 2015; Lee et al, 2016). Methionine sulfoxidation (oxidized methionine, MetO) is recognized to be one such modifi
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