Chaperone Mediated Autophagy Regulates eNOS Uncoupling in Cardiovascular Events

HomeCirculation ResearchVol. 129, No. 10Chaperone Mediated Autophagy Regulates eNOS Uncoupling in Cardiovascular Events Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBChaperone Mediated Autophagy Regulates eNOS Uncoupling in Cardiovascular Events Kathy O. Lui and Yu Huang Kathy O. LuiKathy O. Lui Correspondence to: Kathy O. Lui, PhD, The Chinese University of Hong Kong, Hong Kong, China. Email E-mail Address: [email protected] https://orcid.org/0000-0002-1616-3643 Department of Chemical Pathology, Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China (K.O.L.). Search for more papers by this author and Yu HuangYu Huang https://orcid.org/0000-0002-1277-6784 Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China (Y.H.). Search for more papers by this author Originally published28 Oct 2021https://doi.org/10.1161/CIRCRESAHA.121.320212Circulation Research. 2021;129:946–948This article is a commentary on the followingChaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion InjuryArticle, see p 930eNOS (endothelial NO synthase) has been acknowledged as the major weapon of defense for vascular endothelial cells (ECs) to fight against cardiovascular diseases. Under physiological conditions, eNOS generates the vasoprotective gas molecule NO that regulates vascular tone, local blood flow, and blood pressure. Under pathological situations associated with oxidative stress, eNOS produces superoxide anion at the expense of NO—a process known as eNOS uncoupling that has been attributed to the deficiency of eNOS cofactor tetrahydrobiopterin (BH4), depletion of eNOS substrate L-arginine, and eNOS S-glutathionylation.1 eNOS uncoupling resulting in increased formation of superoxide anion is also a major cause of endothelial dysfunction, particularly in atherosclerosis1 and diabetes.2The synthesis, stability, and activity of eNOS are tightly orchestrated by multiple mechanisms including transcriptional regulation, posttranslational modification such as phosphorylation/dephosphorylation, as well as protein degradation. The ubiquitin-proteasome system has been reported to regulate eNOS coupling possibly through the degradation of BH4.3 When BH4 availability is limited, eNOS no longer produces NO but generates superoxide anion. The rate-limiting enzyme involved in de novo synthesis of BH4, GTPCH (guanosine 5′-triphosphate cyclohydrolase I), is degraded by the 26S proteasome, contributing to BH4 deficiency in diabetic endothelial dysfunction.2 Moreover, endothelial dihydrofolate reductase that catalyzes the regeneration of BH4 from its oxidized form is also regulated by the ubiquitin-proteasome system.4 Nevertheless, BH4 supplementation does not improve NO production or endothelial function in patients with coronary artery disease.5 Alternative strategies targeting the vascular redox state are needed. In addition to ubiquitin-proteasome system, autophagy is another major cellular process for protein degradation. Previous work indicates that autophagy could regulate NO bioavailability.6 In one study, shear stress–induced eNOS phosphorylation and NO production are markedly reduced in autophagy-deficient ECs, whereas production of endothelial reactive oxygen species and inflammatory cytokines is elevated in these cells,7 suggesting a potential contribution of autophagy in maintaining eNOS coupling in ECs. Nonetheless, the exact mechanism by which autophagy regulates eNOS activity especially in cardiovascular events remains elusive.In this issue of Circulation Research, Subramani et al8 demonstrate that eNOS is lost in ECs after hypoxia/reoxygenation (H/R) that can be recovered using a lysosomal pathway inhibitor or various inhibitors of the chaperon-mediated autophagy (CMA) pathway. Although there is a cross talk between CMA and other proteolytic systems for sustaining cell survival,9 the authors show that inhibiting macroautophagy or ubiquitin-proteasome system does not regenerate eNOS. Through high-resolution imaging including immunoelectron microscopy and confocal microscopy, the authors further reveal that eNOS is localized in the autophagolysosome of ECs after H/R and S-glutathionylation of eNOS (SG-eNOS) induces its interaction with LAMP2A (lysosome-associated membrane protein type 2A) via the chaperon HSC70 (heat shock cognate protein of 70 kDa) as depletion of endothelial HSC70 results in localization of SG-eNOS in the cytosol but not the LAMP2A-labeled vesicles. In addition, Cys691 and Cys910 are essential glutathionylated sites in eNOS as mutation on those residues would render eNOS inability to colocalize with LAMP2A. Furthermore, the authors show that prolonged retention of uncoupled eNOS in the cytosol promotes CMA, whereas treatment with the deglutathionylating agent Trx (thioredoxin) regenerates eNOS and inhibits CMA. In addition to Trx, N-acetyl-L-cysteine that promotes deglutathionylation of glutathionylated proteins including SG-eNOS in this case also restores endothelial eNOS expression with reduced CMA. These results suggest that S-glutathionylation of eNOS is required for the initiation of CMA in ECs after H/R.To date, the selectivity of this form of autophagy is conferred by a pentapeptide amino acid KFERQ-like motif in the CMA substrates recognized by HSC70.10 HSC70 then binds to the CMA substrates via this motif to form a chaperon-substrate complex that interacts with LAMP2A at the lysosomal surface11 and translocates into the lysosomal lumen after unfolding assisted by a luminal chaperone (Lys-HSC70) for degradation.12 In this study, Subramani et al8 identified 2 KFERQ-like motifs in the eNOS sequence that are in proximity to Cys691, 679ERLLQ683 and 735QRYRL739. To further confirm that SG-eNOS is a CMA substrate, the authors mutated the KFERQ-like motifs and generated eNOS(L682A, Q683A)-GFP and eNOS(Q735A, R736A)-GFP by site-directed mutagenesis. They find that eNOS(Q735A, R736A) but not eNOS(L682A, Q683A) fails to undergo degradation after H/R and does not interact with HSC70 as demonstrated by coimmunoprecipitation. Moreover, eNOS(Q735A, R736A) but not eNOS(L682A, Q683A) is not able to accumulate in LAMP2A vesicles. Therefore, S-glutathionylation of Cys691 in eNOS during H/R exposes the 735QRYRL739 motif to interact with HSC70 that shuttles SG-eNOS to LAMP2A vesicles for degradation. Nevertheless, a previous study has demonstrated that the molecular chaperon heat shock protein (HSP) 90 can also interact with eNOS and the E3 ubiquitin ligase CHIP (carboxyl terminus of HSC70 interacting protein).13 Instead of being a target for CHIP-mediated ubiquitination and proteasome-dependent degradation, CHIP inactivates eNOS by deviating its trafficking from the Golgi compartment that is required for the eNOS activity.13 Since CHIP can also bind to the molecular chaperon HSC70, whether CHIP is involved in CMA of SG-eNOS and whether there is a cross talk between these two pathways merit future investigations.In vivo, Subramani et al8 also demonstrated that CMA-mediated SG-eNOS degradation is a new mechanism of irreversible loss of eNOS after ischemia-reperfusion injury (I/R). Compared with wild-type controls, ectopic expression of Trx in mice (Trx-Tg) demonstrates significantly reduced levels of SG-eNOS, undiminished levels of eNOS, and the lack of eNOS interaction with HSC70 or LAMP2A as detected in the infarcted area of the I/R heart sections. More importantly, Trx-Tg mice or mice injected with the recombinant human Trx protein at the beginning of I/R also show significant improvement in myocardial perfusion and significantly reduced myocardial apoptosis in the infarcted area. Taken together, these findings have uncovered novel insights into the mechanism behind endothelial dysfunction after H/R or I/R and how eNOS is lost in ECs through S-glutathionylation and CMA (Figure). In fact, CMA is often activated by stress such as prolonged starvation14 or oxidative stress15 in which damaged proteins such as the oxidized ones are selectively removed by CMA. CMA in ECs may serve as a protective mechanism to remove deleterious SG-eNOS and inhibit superoxide generation as a result of eNOS uncoupling. This process, however, contributes to an unwanted side effect that is the irreversible loss of eNOS. Therefore, intervention against SG-eNOS by treatment with recombinant human Trx or other reducing agents would offer therapeutic potential to regenerate eNOS and improve myocardial recovery after I/R.Download figureDownload PowerPointFigure. Chaperon-mediated autophagy (CMA) of S-glutathionylated eNOS (endothelial NO synthase) after ischemia-reperfusion injury. During reperfusion of an ischemic heart, eNOS is S-glutathionylated (SG) by Glutathione S transferase po (GSTP) producing superoxide anion (O2−) instead of NO. Prolonged retention of SG-eNOS promotes its degradation via chaperone-mediated autophagy (CMP) contributing to irreversible loss of eNOS. Specifically, the CMA substrate SG-eNOS interacts with the chaperone protein HSC70 (heat shock cognate protein of 70 kDa) via glutathionylation of the cysteine C691 residue exposing the 735QRYRL739 motif on SG-eNOS to interact with HSC70 in the cytosol. The chaperon-substrate complex is subsequently transported to lysosome via binding with the receptor protein LAMP2A (lysosome-associated membrane protein type 2A). Upon unfolding, CMA substrate proteins can cross the lysosomal membrane assisted by lysosomal HSC70 (Lys-HSC70) where the proteins undergo lysosomal degradation. To prevent the loss of eNOS, deglutathionylation of SG-eNOS by human recombinant thioredoxin (rhTrx) can inhibit CMA and thus restore NO production leading to improved myocardial perfusion and reduced myocardial apoptosis.Article InformationSources of FundingK.O. Lui is supported by the National Natural Science Foundation of China (81922077, 82070494) and Research Grants Council of Hong Kong (14100021, 14108420, M-402-20, and C4026-17WF). Y. Huang is supported by the Research Grants Council of Hong Kong (SRFS2021-4S04, 14164817, and C4024-16 W).DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.For Sources of Funding and Disclosures, see page 948.Correspondence to: Kathy O. Lui, PhD, The Chinese University of Hong Kong, Hong Kong, China. Email [email protected]edu.hkReferences1. Förstermann U, Xia N, Li H. Roles of vascular oxidative stress and nitric oxide in the pathogenesis of atherosclerosis.Circ Res. 2017; 120:713–735. doi: 10.1161/CIRCRESAHA.116.309326LinkGoogle Scholar2. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH. Proteasome-dependent degradation of guanosine 5’-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus.Circulation. 2007; 116:944–953. doi: 10.1161/CIRCULATIONAHA.106.684795LinkGoogle Scholar3. Stangl K, Stangl V. The ubiquitin-proteasome pathway and endothelial (dys)function.Cardiovasc Res. 2010; 85:281–290. doi: 10.1093/cvr/cvp315CrossrefMedlineGoogle Scholar4. Johnston JA, Johnson ES, Waller PR, Varshavsky A. Methotrexate inhibits proteolysis of dihydrofolate reductase by the N-end rule pathway.J Biol Chem. 1995; 270:8172–8178. doi: 10.1074/jbc.270.14.8172CrossrefMedlineGoogle Scholar5. Cunnington C, Van Assche T, Shirodaria C, Kylintireas I, Lindsay AC, Lee JM, Antoniades C, Margaritis M, Lee R, Cerrato R, et al.. Systemic and vascular oxidation limits the efficacy of oral tetrahydrobiopterin treatment in patients with coronary artery disease.Circulation. 2012; 125:1356–1366. doi: 10.1161/CIRCULATIONAHA.111.038919LinkGoogle Scholar6. Nussenzweig SC, Verma S, Finkel T. The role of autophagy in vascular biology.Circ Res. 2015; 116:480–488. doi: 10.1161/CIRCRESAHA.116.303805LinkGoogle Scholar7. Bharath LP, Mueller R, Li Y, Ruan T, Kunz D, Goodrich R, Mills T, Deeter L, Sargsyan A, Anandh Babu PV, et al.. Impairment of autophagy in endothelial cells prevents shear-stress-induced increases in nitric oxide bioavailability.Can J Physiol Pharmacol. 2014; 92:605–612. doi: 10.1139/cjpp-2014-0017CrossrefMedlineGoogle Scholar8. Subramani J, Kundumani-Sridharan V, Das KC. Chaperone-mediated autophagy of enos in myocardial ischemia-reperfusion injury.Circ Res. 2021: 129:930–945. doi: 10.1161/CIRCRESAHA.120.31792LinkGoogle Scholar9. Cuervo AM, Wong E. Chaperone-mediated autophagy: roles in disease and aging.Cell Res. 2014; 24:92–104. doi: 10.1038/cr.2013.153CrossrefMedlineGoogle Scholar10. Dice JF. Peptide sequences that target cytosolic proteins for lysosomal proteolysis.Trends Biochem Sci. 1990; 15:305–309. doi: 10.1016/0968-0004(90)90019-8CrossrefMedlineGoogle Scholar11. Cuervo AM, Dice JF. A receptor for the selective uptake and degradation of proteins by lysosomes.Science. 1996; 273:501–503. doi: 10.1126/science.273.5274.501CrossrefMedlineGoogle Scholar12. Salvador N, Aguado C, Horst M, Knecht E. Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state.J Biol Chem. 2000; 275:27447–27456. doi: 10.1074/jbc.M001394200CrossrefMedlineGoogle Scholar13. Jiang J, Cyr D, Babbitt RW, Sessa WC, Patterson C. Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP.J Biol Chem. 2003; 278:49332–49341. doi: 10.1074/jbc.M304738200CrossrefMedlineGoogle Scholar14. Cuervo AM, Knecht E, Terlecky SR, Dice JF. Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation.Am J Physiol. 1995; 269:C1200–C1208. doi: 10.1152/ajpcell.1995.269.5.C1200CrossrefMedlineGoogle Scholar15. Kiffin R, Christian C, Knecht E, Cuervo AM. Activation of chaperone-mediated autophagy during oxidative stress.Mol Biol Cell. 2004; 15:4829–4840. doi: 10.1091/mbc.e04-06-0477CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesChaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion InjuryJaganathan Subramani, et al. Circulation Research. 2021;129:930-945 October 29, 2021Vol 129, Issue 10 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.121.320212PMID: 34709934 Originally publishedOctober 28, 2021 KeywordsNO synthase type IIIEditorialsautophagyendothelial cellsischemiamolecular chaperonesPDF download Advertisement