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O-Glcnac-Modified SNAP29 Inhibits Autophagy-Mediated Degradation

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O-Glcnac-Modified SNAP29 Inhibits Autophagy-Mediated Degradation 3278 INTERNATIONAL JOURNAL OF MOleCular meDICine 42: 3278-3290, 2018 O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats LIN HUANG1*, PING YUAN1*, PENG YU1, QILING KONG1, ZIXUAN XU1, XIA YAN2, YANG SHEN2,3, JUESHENG YANG4, RONG WAN2, KUI HONG1-3, YANHUA TANG4 and JINZHU HU1 1Department of Cardiovascular Medicine, 2The Jiangxi Key Laboratory of Molecular Medicine, Departments of 3Genetic Medicine and 4Cardiovascular Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China Received March 14, 2018; Accepted August 29, 2018 DOI: 10.3892/ijmm.2018.3866 Abstract. The O-linked β-N-acetylglucosamine (O-GlcNAc) autophagic flux is inhibited, were observed in rats 8 weeks modification and autophagy are associated with diabetic following STZ induction. Subsequently, the selective myocardial injury, however, the molecular mechanisms O-GlcNAcase inhibitor, thiamet G, increased the level of between the two processes remain to be fully elucidated. The O‑GlcNAc modification, which further disrupted autophagic purpose of the present study was to elucidate the molecular flux; deteriorated cardiac diastolic function, as indicated by an regulation of autophagy by O-GlcNAc-modified synapto- increased left ventricular filling peak velocity/atrial contrac- somal-associated protein 29 (SNAP29) in diabetic myocardial tion flow peak velocity ratio shown by echocardiography; and injury. A rat model of type I diabetes was established via exacerbated myocardial abnormalities, as characterized by intraperitoneal injection of streptozotocin (STZ; 55 mg/kg). cardiomyocyte disorganization and fat and interstitial fibrosis Significant increases in the O‑GlcNAc modification and accu- accumulation. By contrast, 6‑diazo‑5‑oxo‑L‑norleucine, an mulation of the autophagy markers microtubule-associated inhibitor of glucosamine fructose-6-phosphate aminotrans- protein 1 light chain 3α II/I and P62, which suggest that ferase isomerizing 1, acted as an O-GlcNAc antagonist and reduced the level of O‑GlcNAc modification, which main- tained autophagic flux and improved cardiac diastolic function. In vitro, high glucose (25 mM) was used to stimulate primary Correspondence to: Dr Jinzhu Hu, Department of Cardiovascular neonatal rat cardiomyocytes (NRCMs). Consistent with the Medicine, The Second Affiliated Hospital of Nanchang University, myocardium of diabetic rats, it was also shown in the NRCMs 1st Minde Road, Nanchang, Jiangxi 330006, P.R. China that O‑GlcNAc modification of SNAP29 negatively regulated E-mail: [email protected] autophagic flux. The application of the short hairpin RNA interference lysosome-associated membrane protein (LAMP2) Dr Yanhua Tang, Department of Cardiovascular Surgery, The Second Affiliated Hospital of Nanchang University, 1st Minde Road, and the autophagy inhibitor 3-methyladenine demonstrated Nanchang, Jiangxi 330006, P.R. China that high glucose inhibited autophagy-mediated degradation E-mail: [email protected] rather than affected the initial stage of autophagy. Finally, co-immunoprecipitation was used to determine the role of *Contributed equally the O-GlcNAc-modified substrate protein SNAP29, which acted as an SNAP29‑syntaxin‑17 (STX17)‑vesicle‑associated Abbreviations: O-GlcNAc, O-linked β-N-acetylglucosamine; membrane protein 8 (VAMP8) complex during disease SNAP29, synaptosomal-associated protein 29; STZ, streptozotocin; progression. The present study is the first, to the best of our TG, thiamet G; Don, 6‑diazo‑5‑oxo‑L‑norleucine; NRCMs, neonatal knowledge, to demonstrate that SNAP29 is an O-GlcNAc rat cardiomyocytes; DM, diabetes mellitus; OGT, O-GlcNAc substrate and that an increase in O‑GlcNAc‑modified SNAP29 transferase; OGA, O-GlcNAcase; TEM, transmission electron inhibits SNAP29‑STX17‑VAMP8 complex formation, thereby microscopy; co‑IP, coimmunoprecipitation; H&E, hematoxylin and eosin; 3-MA, 3-methyladenine; LAMP2, lysosome-associated inhibiting the degradation of autophagy and exacerbating membrane protein 2; LC3, microtubule-associated protein 1 light myocardial injury in type I diabetic rats. chain 3α; VAMP8, vesicle‑associated membrane protein 8; STX17, syntaxin‑17 Introduction Key words: O-linked β-N-acetylglucosamine, autophagy, diabetes The cardiovascular complications of diabetes mellitus (DM), mellitus, myocardial injury, SNAP29 including heart failure and arrhythmia, lead to high rates of disability and mortality (1-3). The molecular mechanism of myocardial injury in DM is complicated, and further HUANG et al: O-GlcNAc-MODIFIED SNAP29 INHIBITS AUTOPHAGY IN TYPE I DIABETES 3279 investigation of this pathogenic mechanism is beneficial to that SNAP29 is involved in mediating membrane fusion further improving the prognosis of DM. between autophagosomes and lysosomes, and forms a complex The posttranslational modification of O-linked with syntaxin‑17 (STX17) and vesicle‑associated membrane β-N-acetylglucosamine (O-GlcNAc) regulates proliferation, protein 8 (VAMP8) to participate in the autophagy process. differentiation, apoptosis, autophagy and other physiological A previous study (23) showed that SNAP29 is modified as and pathological processes in a variety of cells, including a protein substrate by O-GlcNAc in Nematodes. However, cardiomyocytes (4-9). Two genes encode the enzymes for whether the O‑GlcNAc modification of SNAP29 regulates O-GlcNAcylation; O-GlcNAc transferase (OGT) catalyzes autophagic flux in a mammalian disease model remains to be the addition of UDP-GlcNAc to target proteins, whereas elucidated. O-GlcNAcase (OGA) removes the GlcNAc moiety from proteins (10). Accordingly, the selective O-GlcNAcase Materials and methods inhibitor, thiamet G (TG), effectively increases the level of O‑GlcNAc modification. The concerted actions of the two Reagents. The primary antibody mouse anti-O-GlcNAc (RL2; enzymes allow the O‑GlcNAc modification to be a dynamic cat. no. RB229146; 1:1,000) was purchased from Life Company post‑translational modification; 6‑diazo‑5‑oxo‑L‑norleucine (New York, USA). The anti-OGT (cat. no. ab177941; 1:1,000), (Don) is an inhibitor of glucosamine fructose-6-phosphate anti-LAMP2 (cat. no. ab125068 1:1,000), anti-SNAP29 (cat. aminotransferase isomerizing 1, which is an enzyme that no. ab181151; 1:1,000), anti-VAMP8 (cat. no. ab76021; 1:1,000), controls the flux of glucose into the hexosamine pathway and anti‑STX17 (cat. no. ab116113; 1:1,000) and anti‑OGA (cat. catalyzes the formation of glucosamine 6-phosphate. In 2013, no. ab105217; 1:1,000) antibodies were obtained from Abcam a study published in the Journal Nature (11) first applied Don (Cambridge, USA). The anti-Beclin1 (cat. no. D40C5; 1:1,000) as a specific O‑GlcNAc antagonist, which showed that 5 µM antibody was obtained from Cell Signaling Technology, Inc. of Don effectively inhibited the level of O-GlcNAc in vitro. (Danvers, MA, USA). The anti-P62 (cat. no. 18420-1-AP; Several studies have subsequently used Don as an inhibitor of 1:1,000) and anti-Tubulin (cat. no. 66240-1-Ig; 1:1,000) anti- O-GlcNAc. Based on these previous publications (12,13), Don bodies were obtained from ProteinTech Group, Inc. (Chicago, was selected as a selective O-GlcNAc inhibitor in the present IL, USA). Protein A/G beads were obtained from Abmart, study to clarify the role of O‑GlcNAc modified SNAP29 in Inc. (Shanghai, China). Anti-LC3B antibody (cat. no. L7543; cardiomyopathy in type I diabetic rats. Previous studies (11,14) 1:1,000), STZ (cat. no. SD130), TG (cat. no. SML0244), and have found that the acute inhibition of O-GlcNAc inhibits Don (cat. no. D2141) were purchased from Merck KGaA premature ventricular complexes activated by diabetic hyper- (Darmstadt, Germany). The RNAi-LAMP2 sequences were glycemia through O-GlcNAc- and Ca2+/calmodulin-dependent purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, protein kinase II-dependent pathways. Additionally, the dysreg- USA). The three sequence pairs of RNAi-LAMP2 were as ulation of O-GlcNAcylation is likely to be important in the rat follows: Sequence 1 forward, 5'-GAA GUU CUU AUA UGU cardiac mitochondrial dysfunction associated with diabetes, GCA ATT-3' and reverse, 5'-UUG​CAC​AUA UAA GAA​CUU which was reported by Banerjee et al (15). Reportedly (16), CTT-3'; sequence 2 forward, 5'-GGC​AGG AGU ACU UAU UCU an excessive level of O‑GlcNAc modification in a DM animal ATT-3' and reverse, 5'-UAG AAU AAG UAC​UCC​UGC​CTT-3'; model led to the worsening of cardiac function, whereas a sequence 3 forward, 5'‑CUG CAA UCU GAU UGA UUA UU-3' reduction in O-GlcNAc levels improved cardiac function. and reverse, 5'-TAA ACA CTG CTT GAC​CAC​C‑3'. The most However, the mechanism between the O‑GlcNAc modification effective sequence was selected according to the interference and the deterioration of cardiac function remains to be fully efficiency detected using western blot analysis. elucidated. Autophagy is a dynamic process, which is regulated by Animal experiments. All animal experiments were approved multiple genes and molecular signals. The process of autopha- by the Animal Ethics and Experimentation Committee gosome formation, transport of the autophagic substrate to of Nanchang University (Nanchang, China) and were lysosomes, and autophagosomal degradation in lysosomes performed in accordance with the ʻGuide for the Care and is termed autophagic flux. Autophagy is an important meta- Use of Laboratory Animalsʼ (revised 1996). The experimental bolic pathway
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