A FRET biosensor reveals free zinc deficiency in diabetic beta-cell vesicles  

作　　者：Yi Xian Mengxuan Zhou Shangchen Han Rong Yang Yi Wang 

机构地区：[1]College of Life Science and Technology,Huazhong University of Science and Technology,Wuhan 430074,China [2]Peking-Tsinghua Center for Life Sciences,PKU-IDG/McGovern Institute for Brain Research,Academy for Advanced Interdisciplinary Studies,Peking University,Beijing 100871,China [3]State Key Laboratory of Membrane Biology,Beijing Key Laboratory of Cardiometabolic Molecular Medicine,Institute of Molecular Medicine,Peking University,Beijing 100871,China

出　　处：《Chinese Chemical Letters》2020年第2期468-472,共5页中国化学快报（英文版）

基　　金：supported by a China Postdoctoral Science Foundation-funded project(No.2017M612440)。

摘　　要：The concentration of free zinc within insulin-storing vesicles is important for vesicle maturity and therefore requires accurate measurement.However,common small-molecule intensity-based probes and most available genetically encoded Forster resonance energy transfer(FRET)-based sensors for zinc are unsuitable for estimating the free zinc concentration in insulin-storing vesicles.Therefore,we have developed a novel FRET-based zinc sensor based on the RING motif of TRIM72,referred to as ZnT72 R,which has an approximate K_d that varies from 6.07±0.28 μmol/L to 7.84±0.42μmol/L in vitro and a cytosol-calibrated K_d of approximately 55.56±4.59μmol/L in HEK293T cells.To pinpoint the free zinc concentration of insulin-storing vesicles,we initially targeted ZnT72R to beta-cell vesicles by fu sing them to NPY(neuropeptide Y).Following NPY-ZnT72R labeling,the FRET intensity ratios of vesicles were quantified.We found that the free zinc concentration in insulin-storing vesicles of diabetic db/db mice(28.30±1.33μmol/L) was significantly lower than that of control mice(41.46±3.53μmol/L).The concentration of free zinc within insulin-storing vesicles is important for vesicle maturity and therefore requires accurate measurement.However,common small-molecule intensity-based probes and most available genetically encoded Forster resonance energy transfer(FRET)-based sensors for zinc are unsuitable for estimating the free zinc concentration in insulin-storing vesicles.Therefore,we have developed a novel FRET-based zinc sensor based on the RING motif of TRIM72,referred to as ZnT72 R,which has an approximate K_d that varies from 6.07±0.28 μmol/L to 7.84±0.42μmol/L in vitro and a cytosol-calibrated K_d of approximately 55.56±4.59μmol/L in HEK293T cells.To pinpoint the free zinc concentration of insulin-storing vesicles,we initially targeted ZnT72R to beta-cell vesicles by fu sing them to NPY(neuropeptide Y).Following NPY-ZnT72R labeling,the FRET intensity ratios of vesicles were quantified.We found that the free zinc concentration in insulin-storing vesicles of diabetic db/db mice(28.30±1.33μmol/L) was significantly lower than that of control mice(41.46±3.53μmol/L).

关 键 词：Zinc FRET Insulin BIOSENSOR Beta cell VESICLE Diabetes 

分 类 号：O657.3[理学—分析化学] R587.1[理学—化学]