为探究脯氨酰内肽酶(prolyl endopeptidase,PEP)在骨胶原蛋白肽酶法制备过程中的应用潜力,分别以牛骨胶原蛋白(bovine bone collagen,BBC)、猪骨胶原蛋白(porcine bone collagen,PBC)、鸡骨胶原蛋白(chicken bone collagen,CBC)为原料,...为探究脯氨酰内肽酶(prolyl endopeptidase,PEP)在骨胶原蛋白肽酶法制备过程中的应用潜力,分别以牛骨胶原蛋白(bovine bone collagen,BBC)、猪骨胶原蛋白(porcine bone collagen,PBC)、鸡骨胶原蛋白(chicken bone collagen,CBC)为原料,对比分析3种不同来源骨胶原蛋白的序列特征,预测不同骨胶原蛋白潜在的酶解作用位点和理论水解度。在55℃、pH 8.0条件下,利用PEP水解处理3种不同来源骨胶原蛋白,通过水解度、分子质量分布测定和扫描电子显微镜观察等方法进行表征。利用红外光谱、X射线衍射和圆二色光谱等方法探究酶解过程中胶原蛋白结构变化。结果表明,PEP对3种不同来源骨胶原蛋白均有显著水解效果,PBC的水解度最高,为51.35%,其次分别为CBC(29.81%)和BBC(22.81%)。3种骨胶原蛋白酶解产物分子质量多分布在500 Da以下。光谱学分析表明PEP破坏了胶原蛋白的三螺旋结构,从而使胶原蛋白降解,达到制备胶原蛋白肽的效果。PEP可以高效酶解骨胶原蛋白制备小分子蛋白肽,本研究可为功能性骨胶原蛋白肽的酶法制备提供依据。展开更多
Background and Aims:Prolyl endopeptidase(PREP)is a serine endopeptidase that participates in many pathological processes including inflammation,oxidative stress,and autophagy.Our previous studies found that PREP knock...Background and Aims:Prolyl endopeptidase(PREP)is a serine endopeptidase that participates in many pathological processes including inflammation,oxidative stress,and autophagy.Our previous studies found that PREP knockout exhibited multiple benefits in high-fat diet(HFD)or methionine choline-deficient diet-induced metabolic dysfunctionassociated fatty liver disease(MAFLD).However,cumulative studies have suggested that PREP performs complex functions during disease development.Therefore,further understanding the role of PREP in MAFLD development is the foundation of PREP intervention.Methods:In this study,an HFD-induced MAFLD model at different time points(4,8,12,and 16 weeks)was used to explore dynamic changes in the PREP proline-glycine-proline(PGP)/N-acetyl-seryl-aspartyllysyl-proline(AcSDKP)system.To explore its potential value in MAFLD treatment,saline,or the PREP inhibitor,KYP-2047,was administered to HFD-induced MAFLD mice from the 10th to 16th weeks.Results:PREP activity and expression were increased in HFD-mice compared with control mice from the 12th week onwards,and increased PREP mainly resulted in the activation of the matrix metalloproteinase 8/9(MMP8/9)-PREP-PGP axis rather than the thymosin B4-meprin a/PREP-AcSDKP axis.In addition,KYP-2047 reduced HFD-induced liver injury and oxidative stress,improved lipid metabolism through the suppression of lipogenic genes and the induction of B-oxidation-related genes,and attenuated hepatic inflam-mation by decreasing MMP8/9 and PGP.Moreover,KYP2047 restored HFD-induced impaired autophagy and this was veri-fied in HepG2 cells.Conclusions:These findings suggest that increased PREP activity/expression during MAFLD de-velopment might be a key factor in the transition from sim-ple steatosis to steatohepatitis,and KYP-2047 might possess therapeutic potential for MAFLD treatment.展开更多
文摘为探究脯氨酰内肽酶(prolyl endopeptidase,PEP)在骨胶原蛋白肽酶法制备过程中的应用潜力,分别以牛骨胶原蛋白(bovine bone collagen,BBC)、猪骨胶原蛋白(porcine bone collagen,PBC)、鸡骨胶原蛋白(chicken bone collagen,CBC)为原料,对比分析3种不同来源骨胶原蛋白的序列特征,预测不同骨胶原蛋白潜在的酶解作用位点和理论水解度。在55℃、pH 8.0条件下,利用PEP水解处理3种不同来源骨胶原蛋白,通过水解度、分子质量分布测定和扫描电子显微镜观察等方法进行表征。利用红外光谱、X射线衍射和圆二色光谱等方法探究酶解过程中胶原蛋白结构变化。结果表明,PEP对3种不同来源骨胶原蛋白均有显著水解效果,PBC的水解度最高,为51.35%,其次分别为CBC(29.81%)和BBC(22.81%)。3种骨胶原蛋白酶解产物分子质量多分布在500 Da以下。光谱学分析表明PEP破坏了胶原蛋白的三螺旋结构,从而使胶原蛋白降解,达到制备胶原蛋白肽的效果。PEP可以高效酶解骨胶原蛋白制备小分子蛋白肽,本研究可为功能性骨胶原蛋白肽的酶法制备提供依据。
基金supported by grants from the National Natural Science Foundation of China(81970511,82270620).
文摘Background and Aims:Prolyl endopeptidase(PREP)is a serine endopeptidase that participates in many pathological processes including inflammation,oxidative stress,and autophagy.Our previous studies found that PREP knockout exhibited multiple benefits in high-fat diet(HFD)or methionine choline-deficient diet-induced metabolic dysfunctionassociated fatty liver disease(MAFLD).However,cumulative studies have suggested that PREP performs complex functions during disease development.Therefore,further understanding the role of PREP in MAFLD development is the foundation of PREP intervention.Methods:In this study,an HFD-induced MAFLD model at different time points(4,8,12,and 16 weeks)was used to explore dynamic changes in the PREP proline-glycine-proline(PGP)/N-acetyl-seryl-aspartyllysyl-proline(AcSDKP)system.To explore its potential value in MAFLD treatment,saline,or the PREP inhibitor,KYP-2047,was administered to HFD-induced MAFLD mice from the 10th to 16th weeks.Results:PREP activity and expression were increased in HFD-mice compared with control mice from the 12th week onwards,and increased PREP mainly resulted in the activation of the matrix metalloproteinase 8/9(MMP8/9)-PREP-PGP axis rather than the thymosin B4-meprin a/PREP-AcSDKP axis.In addition,KYP-2047 reduced HFD-induced liver injury and oxidative stress,improved lipid metabolism through the suppression of lipogenic genes and the induction of B-oxidation-related genes,and attenuated hepatic inflam-mation by decreasing MMP8/9 and PGP.Moreover,KYP2047 restored HFD-induced impaired autophagy and this was veri-fied in HepG2 cells.Conclusions:These findings suggest that increased PREP activity/expression during MAFLD de-velopment might be a key factor in the transition from sim-ple steatosis to steatohepatitis,and KYP-2047 might possess therapeutic potential for MAFLD treatment.
文摘目的观察高脂饮食诱导的非酒精性脂肪性肝病(NAFLD)小鼠肝脏脯氨酰内肽酶(PREP)蛋白水平及活性的变化。方法随机将40只雄性C57BL/6J小鼠分成普通饲料和高脂饲料喂养组,分别在喂养4 w和16 w处死小鼠。检测血生化指标,观察肝组织病理学变化并计算NAFLD活动度积分(NAS);采用荧光定量PCR法、Western blot法和Suc-Gly-Pro-AMC底物法分别检测肝组织PREP m RNA、蛋白水平和活性变化。结果在高脂饮食喂养4 w,模型组小鼠肝细胞出现轻度脂肪变,NAS积分为(3.43±0.79),较对照组显著升高;在高脂饮食喂养16w,模型组小鼠体质量、肝指数、ALT、甘油三脂、总胆固醇和空腹血糖分别为(32.93±2.72)g、(4.64±0.46)%、(53.64±22.01)IU/L、(1.18±0.17)mmol/L、(4.34±0.44)mmol/L和(5.67±0.87)mmol/L,显著高于对照组【分别为(28.19±1.23)g、(4.02±0.11)%、(28.60±4.01)IU/L、(0.99±0.15)mmol/L、(2.94±0.18)mmol/L和(4.26±0.65)mmol/L,P<0.05】;模型组肝组织NAS积分为(5.60±1.43),显著高于对照组【(0.40±0.70),P<0.01】;PREP蛋白水平与对照组相比无显著性差异,但PREP m RNA水平和酶活性分别为对照组的(1.81±0.34)倍(P<0.01)和(1.36±1.78)倍(P<0.05)。结论小鼠肝组织PREP活性变化可能参与了小鼠非酒精性单纯性脂肪肝向脂肪性肝炎的转变。
