决明1-脱氧-D-木酮糖-5-磷酸合酶和1-脱氧-D-木酮糖-5-磷酸还原异构酶的功能鉴定与表达分析

本研究根据决明三代转录组测序获得的1-脱氧-D-木酮糖-5-磷酸合酶(CoDXS)和1-脱氧-D-木酮糖-5-磷酸还原异构酶(CoDXR)基因的全长序列设计特异性引物,PCR扩增获得CoDXS、CoDXR基因的OFR,构建原核表达载体pTrc-CoDXS、pTrc-CoDXR在含有质粒PAC-BETA的TOP10重组菌中进行表达,通过TOP10的菌斑颜色变化来验证CoDXS和CoDXR基因的功能。利用实时荧光定量PCR来检测CoDXS、CoDXR基因在决明不同组织中以及六种胁迫条件下的表达情况。研究结果表明CoDXS、CoDXR基因的OFR分别为2127、1416 bp,编码的氨基酸数目分别为708、471,功能鉴定结果显示含有CoDXS、CoDXR基因编码区的TOP10菌斑呈现出深橘黄色;CoDXS基因的表达趋势为:花>茎>种子>叶>根,CoDXR基因的表达趋势为:叶>花>茎>根>种子。对CoDXR和CoDXS基因在不同的胁迫条件下的相对表达量检测分析发现,在盐胁迫条件下:CoDXS基因的表达量整体呈现出下降趋势,CoDXR基因的表达量波动范围较小;在ABA胁迫条件下:CoDXS基因的表达量呈现出先下降上升趋势,CoDXR基因的表达量呈现出上升趋势;在MeJA胁迫条件下:CoDXR和CoDXS基因的表达量均呈现出先下降后上升的趋势;在PEG6000胁迫条件下:对CoDXS基因表达量的影响较小,CoDXR基因的表达量呈现出上升趋势;在高温胁迫条件:CoDXS基因呈现出下降趋势,对CoDXR基因的表达影响较小;低温胁迫条件下:CoDXR和CoDXS基因的表达均呈现出下降趋势。

Infant cholestasis patient with a novel missense mutation in the AKR1D1 gene successfully treated by early adequate supplementation with chenodeoxycholic acid: A case report and review of the literature

Steroid 5β-reductase [aldo-keto reductase family 1 member D1(AKR1D1)] is essential for bile acid biosynthesis. Bile acid deficiency caused by genetic defects in AKR1D1 leads to life-threatening neonatal hepatitis and cholestasis. There is still limited experience regarding the treatment of this disease. We describe an infant who presented with hyperbilirubinemia and coagulopathy but normal bile acid and γ-glutamyltransferase. Gene analysis was performed using genomic DNA from peripheral lymphocytes from the patient, his parents, and his elder brother. The patient was compound heterozygous for c.919C>T in exon 8 and exhibited a loss of heterozygosity of the AKR1D1 gene, which led to an amino acid substitution of arginine by cysteine at amino acid position 307(p.R307C). Based on these mutations, the patient was confirmed to have primary 5β-reductase deficiency. Ursodeoxycholic acid(UDCA) treatment did not have any effect on the patient. However, when we changed to chenodeoxycholic acid(CDCA) treatment, his symptoms and laboratory tests gradually improved. It is therefore crucial to supplement with an adequate dose of CDCA early to improve clinical symptoms and to normalize laboratory tests.

Berberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells

AIdo-keto reductase family 1 member C3 has recently been regarded as a potential therapeutic target in castrate-resistant prostate cancer. Herein, we investigated whether berberine delayed the progression of castrate-resistant prostate cancer by reducing androgen synthesis through the inhibition of Aldo-keto reductase family 1 member C3. Cell viability and cellular testosterone content were measured in prostate cancer cells. Aido-keto reductase family 1 member C3 mRNA and protein level were detected by RT-PCR and Western bolt analyses, respectively. Computer analysis with AutoDock Tools explored the molecular interaction of berberine with Aldo-keto reductase family 1 member C3. We found that berberine inhibited 22Rvl cells proliferation and decreased cellular testosterone formation in a dose-dependent manner. Berberine inhibited Aldo-keto reductase family I member C3 enzyme activity, rather than influenced mRNA and protein expressions. Molecular docking study demonstrated that berberine could enter the active center of Aldo-keto reductase family 1 member C3 and form π-π interaction with the amino-acid residue Phe306 and Phe311. In conclusion, the structural interaction of berberine with Aldo-keto reductase family 1 member C3 is attributed to the suppression of Aldo-keto reductase family I member C3 enzyme activity and the inhibition of 22Rvl prostate cancer cell growth by decreasing the intfacellular androgen synthesis. Our result provides the experimental basis for the design, research, and development of AKRlC3 inhibitors using berberine as the lead compound.

Study of Aldo-keto Reductase 1C3 Inhibitor with Novel Framework for Treating Leukaemia Based on Virtual Screening and In vitro Biological Activity Testing

Aldo-keto reductase 1C3(AKR1C3)is a potential target for the treatment of acute myeloid leukaemia and T-cell acute lymphoblastic leukaemia.In this study,pharmacophore models,molecular docking and virtual screening of target prediction were used to find a potential AKR1C3 inhibitor.Firstly,eight bacteriocin derivatives(Z1-Z8)were selected as training sets to construct 20 pharmacophore models.The best pharmacophore model MODEL_016 was obtained by Decoy test(the enrichment degree was 21.5117,and the fitting optimisation degree was 0.9668).Secondly,MODEL_016 was used for the virtual screening of ZINC database.Thirdly,the hit 83256 molecules were docked into the AKR1C3 protein.Compared to the total scores and interactions between compounds and protein,16532 candidate compounds with higher docking scores and interactions with important residues PHE306 and TRP227 were screened.Lastly,eight compounds(A1-A8)that had good absorption,distribution,metabolism,excretion and toxicity(ADMET)properties were obtained by target prediction.Compounds A3 and A7 with high total score and good target prediction results were selected for in vitro biological activity test,whose IC_(50) values were 268.3 and 88.94µmol/L,respectively.The results provide an important foundation for the discovery of novel AKR1C3 inhibitors.The research methods used in this study can also provide important references for the research and development of new drugs.

东北雷公藤DXR HMGR基因克隆及生物信息学分析

目的:克隆东北雷公藤萜类物质生物合成关键酶基因3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase,HMGR)基因和1-脱氧-D-木酮糖-5-磷酸还原异构酶(1-deoxy-D-xylulose-5-phosphate reductoisomerase,DXR)基因全长,并对其进行生物信息学分析。方法:根据东北雷公藤转录组数据,设计特异性引物,采用聚合酶链式反应(polymerase chain reaction,PCR)技术克隆东北雷公藤DXR、HMGR基因编码区的全长序列,并进行一系列生物信息学分析。结果:成功克隆东北雷公藤DXR、HMGR基因各1条,分别命名为TrDXR和TrHMGR,两者分别长1410、1770 bp,编码469个和589个氨基酸。TrDXR为亲水性蛋白,亚细胞定位在叶绿体,无跨膜结构,转运肽定位于叶绿体,含有2个结合功能域和2个活性位点基序;TrHMGR为疏水性蛋白,亚细胞定位在内质网,有跨膜结构,无转运肽,含有4个结合功能域。TrDXR和TrHMGR与不同物种同源序列的相似性高,保守性强。结论:克隆获得TrDXR、TrHMGR基因cDNA全长,并对其生物功能进行了初步预测,为基因功能鉴定及东北雷公藤萜类物质分子形成机制研究奠定基础。

AKR1B1 promotes pancreatic cancer metastasis by regulating lysosome-guided exosome secretion

Pancreatic cancer is one of the most lethal neoplasms with high metastatic potential and is resistant to almost all current therapies.Epalrestat is an aldo-keto reductase family 1 member B1(AKR1B1)inhibitor for the treatment of diabetic neuropathy,but its potential application in cancer treatment and the underlying mechanism are largely unknown.Here,we found that AKR1B1 is upregulated in pancreatic cancer and is positively associated with metastasis.Upregulated AKR1B1 promoted exosome secretion,accelerating cell migration in pancreatic cancer cells.Further analysis indicated that AKR1B1 negatively regulated lysosomal function and multivesicular body(MVB)degradation in lysosomes.However,AKR1B1 had a minimal role in the generation of MVBs.Transcription factor EB(TFEB)and MVB-expressed RAB7A were two molecular targets that are negatively regulated by AKR1B1.These results uncovered a critical role for AKR1B1 in the regulation of lysosomal function and exosome secretion.Pharmacological targeting of AKR1B1 by clinically used medicines,such as Epalrestat,might represent an efficient way to inhibit pancreatic growth and metastasis.

活性氧在密旋链霉菌Act12诱导丹参毛状根中丹参酮积累中的作用

作者前期研究成果显示密旋链霉菌Act12可以上调丹参酮生物合成途径上的关键酶基因的表达大幅提高丹参毛状根中丹参酮含量。该实验则进一步研究了活性氧在Act12诱导丹参毛状根中丹参酮积累的作用。在继代培养21 d的丹参毛状根中添加Act12不同的诱导子及诱导子组合,分别测定不同收获期毛状根的生物量,毛状根中活性氧积累量,丹参酮类成分的积累量和关键酶基因表达量。Act12诱导后丹参毛状根中活性氧含量上升,HMGR和DXR基因表达上调,最高分别达到对照的32.4,4.8倍,毛状根中丹参酮积累增加,最高达到对照的10.2倍;加入活性氧抑制剂CAT和SOD后,丹参毛状根中的活性氧含量较Act12处理显著下降,HMGR和DXR基因表达也明显下降,毛状根中丹参酮含量较Act12处理下降了74.6%。活性氧介导了Act12诱导丹参酮积累,Act12很可能是通过ROS信号通路激活了MVA和MEP途径,从而提高了丹参酮在毛状根中的含量。

蓝桉叶片桉叶素生物合成机理研究

为探明蓝桉(Eucalyptus globulus Labill.)叶片桉叶素合成过程中关键酶活性与桉叶素含量的关系,以不同发育阶段的蓝桉叶片为研究对象,测定桉叶素相对含量和3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)、1-脱氧-D-木酮糖-5-磷酸合酶(DXS)、异戊烯基焦磷酸异构酶(IPPI)、香叶基焦磷酸合酶(GPPS)、桉叶素合酶(CinS)活性,并对其进行方差分析、相关性分析、通径分析.结果表明,随着蓝桉叶片成熟度增加,桉叶素相对含量逐渐增加,HMGR、DXS、GPPS、IPPI的酶活性逐渐下降.在一个完整的年生长周期内,桉叶素相对含量在7月份显著高于其余月份,HMGR、DXS、GPPS、CinS活性在5、6月份显著高于其余月份.桉叶素相对含量与DXS、CinS活性显著正相关,与HMGR、IPPI、GPPS酶活性极显著负相关.研究结果表明,在5、6月份增强蓝桉幼嫩叶片的DXS和CinS活性,可以有效提高桉叶素积累,进而提高蓝桉桉叶油的产量和品质.

Intracrine androgen biosynthesis and drug resistance

Castration-resistant prostate cancer is the lethal form of prostate cancer and most commonly remains dependent on androgen receptor(AR)signaling.Current therapies use AR signaling inhibitors(ARSI)exemplified by abiraterone acetate,a P450c17 inhibitor,and enzalutamide,a potent AR antagonist.However,drug resistance to these agents occurs within 12-18 months and they only prolong overall survival by 3-4 months.Multiple mechanisms can contribute to ARSI drug resistance.These mechanisms can include but are not limited to germline mutations in the AR,post-transcriptional alterations in AR structure,and adaptive expression of genes involved in the intracrine biosynthesis and metabolism of androgens within the tumor.This review focuses on intracrine androgen biosynthesis,how this can contribute to ARSI drug resistance,and therapeutic strategies that can be used to surmount these resistance mechanisms.