BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associ...BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14(MODY14).Currently,only two mutations[c.1655T>A(p.Leu552*)and c.281G>A p.(Asp94Asn)]have been identified in association with this disease.Given the limited understanding of MODY14,it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations.AIM To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain.METHODS Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study.Whole exome sequencing was performed on the patients as well as their family members.The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis.In addition,the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments.Finally,the impact of these variants on APPL1 protein expression and the insulin pathway were assessed,and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored.RESULTS A total of five novel mutations were identified,including four missense mutations(Asp632Tyr,Arg633His,Arg532Gln,and Ile642Met)and one intronic mutation(1153-16A>T).Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions.The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster.In addition,multiple alignment of amino acid sequences showed that the Arg532Gln,Asp632Tyr,and Arg633His variants were conserved across different species.Moreover,in in vitro functional experiments,both the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels,indicating their pathogenic nature.Therefore,based on the patient’s clinical and family history,combined with the results from bioinformatics analysis and functional experiment,the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were classified as pathogenic mutations.Importantly,all these mutations were located within the phosphotyrosinebinding domain of APPL1,which plays a critical role in the insulin sensitization effect.CONCLUSION This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.展开更多
目的·探究衔接子相关蛋白激酶1(adaptor-associated protein kinase 1,AAK1)新的相互作用蛋白,以及除网格蛋白介导的内吞作用外AAK1介导的生物学功能。方法·通过在HEK-293T细胞中分别外源性转染带有标签的AAK1载体与空白对照...目的·探究衔接子相关蛋白激酶1(adaptor-associated protein kinase 1,AAK1)新的相互作用蛋白,以及除网格蛋白介导的内吞作用外AAK1介导的生物学功能。方法·通过在HEK-293T细胞中分别外源性转染带有标签的AAK1载体与空白对照载体,利用标签特异性的琼脂糖凝胶进行免疫共沉淀(co-immunoprecipitation,CoIP),并联合质谱分析的方法获得潜在与AAK1相互作用的蛋白;通过CoIP初步验证质谱结果;通过荧光共聚焦成像观察AAK1与其潜在结合蛋白在细胞内的空间定位;通过体外纯化重组蛋白,利用谷胱甘肽巯基转移酶融合蛋白沉降实验(glutathione-S-transferase pulldown,GST Pulldown)进一步明确蛋白间是否为直接的相互作用;通过嘌呤霉素结合实验观察AAK1对于细胞内整体翻译水平的调控作用。结果·质谱结果提示AAK1可能与以脆性X相关蛋白1(fragile X mental retardation syndrome-related protein 1,FXR1)、FXR2、脆性X智力低下蛋白(fragile X mental retardation protein 1,FMRP)三者为核心的一系列蛋白形成复合体。外源性转染AAK1-3xFLAG及FMRP-MYC质粒,利用抗FLAG琼脂糖凝胶富集AAK1-3xFLAG后,可以检测到FMRPMYC的表达;利用内源性抗体进行CoIP,发现富集AAK1可以检测到FMRP的表达。荧光共聚焦成像显示EGFP-AAK1与mCherry-FMRP在细胞质中存在部分空间共定位。GST Pulldown显示FMRP可以直接沉淀HIS6-AAK1重组蛋白。嘌呤霉素结合实验显示相同时间内嘌呤霉素标记的细胞内新合成肽段数量与AAK1蛋白表达量呈正相关。结论·AAK1与FMRP在细胞质内存在直接的相互作用,且AAK1可以提高细胞内的翻译水平。展开更多
Evidence suggests that rapid changes to supporting glia may predispose individuals with spinal cord injury(SCI) to such comorbidities. Here, we interrogated the expression of astrocyte-and microglial-specific markers ...Evidence suggests that rapid changes to supporting glia may predispose individuals with spinal cord injury(SCI) to such comorbidities. Here, we interrogated the expression of astrocyte-and microglial-specific markers glial fibrillary acidic protein(GFAP) and ionized calcium binding adaptor molecule 1(Iba1) in the rat brain in the first 24 hours following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy;half of the rats received a mild contusion injury at the level of the T10 vertebral body(SCI group), the other half did not(Sham group). Twenty-four hours post-surgery the amygdala, periaqueductal grey, prefrontal cortex, hypothalamus, lateral thalamus, hippocampus(dorsal and ventral) in rats were collected. GFAP and Iba1 m RNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, GFAP m RNA and protein expression increased in the amygdala and hypothalamus. In contrast, gene and protein expression decreased in the thalamus and dorsal hippocampus. Interestingly, Iba1 transcripts and proteins were significantly diminished only in the dorsal and ventral hippocampus, where gene expression diminished. These findings demonstrate that as early as 24 hours post-SCI there are region-specific disruptions of GFAP and Iba1 transcript and protein levels in higher brain regions. All procedures were approved by the University of Technology Sydney Institutional Animal Care and Ethics Committee(UTS ACEC13-0069).展开更多
目的：探讨中国汉族人群一氧化氮合酶-1接头蛋白（NOS1AP）基因多态性与阿尔茨海默病（AD）的相关性。方法：采用Taq Man探针基因分型技术对300例AD患者（病例组）及716名正常对照者（正常对照组）进行NOS1AP基因多态性（rs1415263、rs4...目的：探讨中国汉族人群一氧化氮合酶-1接头蛋白（NOS1AP）基因多态性与阿尔茨海默病（AD）的相关性。方法：采用Taq Man探针基因分型技术对300例AD患者（病例组）及716名正常对照者（正常对照组）进行NOS1AP基因多态性（rs1415263、rs4145621、rs12742393、rs348624）分型,应用SHEsisplus和SNPstats在线软件比较两组间的等位基因频率、基因型频率、单体型频率分布。结果：rs4145621、rs12742393和rs348624等位基因频率（χ^2=2.78,P=0.095;χ^2=2.489,P=0.115;χ^2=0.002,P=0.964）两组间分布差异无统计学意义;基因型频率在5种遗传模式下（经年龄、性别校正后P均〉0.05）两组间分布差异无统计学意义;单体型CC（rs4145621-rs12742393）正常对照组的频率低于病例组（0.011 vs 0.028;χ^2=6.103,P=0.013）,经性别、年龄校正后差异无统计学意义（P=0.52）,其余CA、TC、TA单体型在两组中分布差异无统计学意义（P〉0.05）。结论：在中国汉族人群中,NOS1AP基因可能不是AD的易感基因。展开更多
Pollen development is a,pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fu...Pollen development is a,pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 （AP1G） mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development.展开更多
家族性高胆固醇血症(familial hypercholesterolemia,FH)的临床特征为血总胆固醇升高,尤其是低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-c)升高,沉积于组织,形成皮肤或肌腱黄色瘤,导致动脉粥样硬化甚至早发冠心病。F...家族性高胆固醇血症(familial hypercholesterolemia,FH)的临床特征为血总胆固醇升高,尤其是低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-c)升高,沉积于组织,形成皮肤或肌腱黄色瘤,导致动脉粥样硬化甚至早发冠心病。FH的发病机制为LDL受体(LDL receptor,LDLR)或apoB基因突变引起LDL受体途径功能缺陷,主要为常染色体显性遗传疾患,具有基因剂量效应;部分患者为常染色体隐性遗传,机制为LDL受体衔接蛋白1(LDL receptor adaptor protein 1,LDLRAP1)失功能型突变,导致LDL内化活性降低。罕见的人类枯草溶菌素转化酶9(proprotein convertase subtilisin/kexin type 9,PCSK9)发生功能型突变也可引起严重的FH表型。PCSK9通过降解LDLR蛋白间接下调LDL受体途径,其失功能突变可致血浆LDL水平下降。因此PCSK9是目前降脂药物的研究热点。展开更多
基金Supported by the National Natural Science Foundation,No.81974124and Taishan Scholar Project,No.tsqn20161071.
文摘BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14(MODY14).Currently,only two mutations[c.1655T>A(p.Leu552*)and c.281G>A p.(Asp94Asn)]have been identified in association with this disease.Given the limited understanding of MODY14,it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations.AIM To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain.METHODS Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study.Whole exome sequencing was performed on the patients as well as their family members.The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis.In addition,the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments.Finally,the impact of these variants on APPL1 protein expression and the insulin pathway were assessed,and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored.RESULTS A total of five novel mutations were identified,including four missense mutations(Asp632Tyr,Arg633His,Arg532Gln,and Ile642Met)and one intronic mutation(1153-16A>T).Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions.The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster.In addition,multiple alignment of amino acid sequences showed that the Arg532Gln,Asp632Tyr,and Arg633His variants were conserved across different species.Moreover,in in vitro functional experiments,both the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels,indicating their pathogenic nature.Therefore,based on the patient’s clinical and family history,combined with the results from bioinformatics analysis and functional experiment,the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were classified as pathogenic mutations.Importantly,all these mutations were located within the phosphotyrosinebinding domain of APPL1,which plays a critical role in the insulin sensitization effect.CONCLUSION This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.
文摘目的·探究衔接子相关蛋白激酶1(adaptor-associated protein kinase 1,AAK1)新的相互作用蛋白,以及除网格蛋白介导的内吞作用外AAK1介导的生物学功能。方法·通过在HEK-293T细胞中分别外源性转染带有标签的AAK1载体与空白对照载体,利用标签特异性的琼脂糖凝胶进行免疫共沉淀(co-immunoprecipitation,CoIP),并联合质谱分析的方法获得潜在与AAK1相互作用的蛋白;通过CoIP初步验证质谱结果;通过荧光共聚焦成像观察AAK1与其潜在结合蛋白在细胞内的空间定位;通过体外纯化重组蛋白,利用谷胱甘肽巯基转移酶融合蛋白沉降实验(glutathione-S-transferase pulldown,GST Pulldown)进一步明确蛋白间是否为直接的相互作用;通过嘌呤霉素结合实验观察AAK1对于细胞内整体翻译水平的调控作用。结果·质谱结果提示AAK1可能与以脆性X相关蛋白1(fragile X mental retardation syndrome-related protein 1,FXR1)、FXR2、脆性X智力低下蛋白(fragile X mental retardation protein 1,FMRP)三者为核心的一系列蛋白形成复合体。外源性转染AAK1-3xFLAG及FMRP-MYC质粒,利用抗FLAG琼脂糖凝胶富集AAK1-3xFLAG后,可以检测到FMRPMYC的表达;利用内源性抗体进行CoIP,发现富集AAK1可以检测到FMRP的表达。荧光共聚焦成像显示EGFP-AAK1与mCherry-FMRP在细胞质中存在部分空间共定位。GST Pulldown显示FMRP可以直接沉淀HIS6-AAK1重组蛋白。嘌呤霉素结合实验显示相同时间内嘌呤霉素标记的细胞内新合成肽段数量与AAK1蛋白表达量呈正相关。结论·AAK1与FMRP在细胞质内存在直接的相互作用,且AAK1可以提高细胞内的翻译水平。
基金The University of Technology Sydney(UTS)Start-up Research Grant 2018 funded this study(to AC)。
文摘Evidence suggests that rapid changes to supporting glia may predispose individuals with spinal cord injury(SCI) to such comorbidities. Here, we interrogated the expression of astrocyte-and microglial-specific markers glial fibrillary acidic protein(GFAP) and ionized calcium binding adaptor molecule 1(Iba1) in the rat brain in the first 24 hours following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy;half of the rats received a mild contusion injury at the level of the T10 vertebral body(SCI group), the other half did not(Sham group). Twenty-four hours post-surgery the amygdala, periaqueductal grey, prefrontal cortex, hypothalamus, lateral thalamus, hippocampus(dorsal and ventral) in rats were collected. GFAP and Iba1 m RNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, GFAP m RNA and protein expression increased in the amygdala and hypothalamus. In contrast, gene and protein expression decreased in the thalamus and dorsal hippocampus. Interestingly, Iba1 transcripts and proteins were significantly diminished only in the dorsal and ventral hippocampus, where gene expression diminished. These findings demonstrate that as early as 24 hours post-SCI there are region-specific disruptions of GFAP and Iba1 transcript and protein levels in higher brain regions. All procedures were approved by the University of Technology Sydney Institutional Animal Care and Ethics Committee(UTS ACEC13-0069).
文摘目的：探讨中国汉族人群一氧化氮合酶-1接头蛋白（NOS1AP）基因多态性与阿尔茨海默病（AD）的相关性。方法：采用Taq Man探针基因分型技术对300例AD患者（病例组）及716名正常对照者（正常对照组）进行NOS1AP基因多态性（rs1415263、rs4145621、rs12742393、rs348624）分型,应用SHEsisplus和SNPstats在线软件比较两组间的等位基因频率、基因型频率、单体型频率分布。结果：rs4145621、rs12742393和rs348624等位基因频率（χ^2=2.78,P=0.095;χ^2=2.489,P=0.115;χ^2=0.002,P=0.964）两组间分布差异无统计学意义;基因型频率在5种遗传模式下（经年龄、性别校正后P均〉0.05）两组间分布差异无统计学意义;单体型CC（rs4145621-rs12742393）正常对照组的频率低于病例组（0.011 vs 0.028;χ^2=6.103,P=0.013）,经性别、年龄校正后差异无统计学意义（P=0.52）,其余CA、TC、TA单体型在两组中分布差异无统计学意义（P〉0.05）。结论：在中国汉族人群中,NOS1AP基因可能不是AD的易感基因。
基金supported by Major Research Plan(2013CB945102)from the Ministry of Science,Technology of ChinaNational Natural Science Foundation of China(31625003 and 31471304 to Y.Z.)partially supported by Tai-Shan Scholar Program by Shandong Provincial Government
文摘Pollen development is a,pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 （AP1G） mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development.
文摘家族性高胆固醇血症(familial hypercholesterolemia,FH)的临床特征为血总胆固醇升高,尤其是低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-c)升高,沉积于组织,形成皮肤或肌腱黄色瘤,导致动脉粥样硬化甚至早发冠心病。FH的发病机制为LDL受体(LDL receptor,LDLR)或apoB基因突变引起LDL受体途径功能缺陷,主要为常染色体显性遗传疾患,具有基因剂量效应;部分患者为常染色体隐性遗传,机制为LDL受体衔接蛋白1(LDL receptor adaptor protein 1,LDLRAP1)失功能型突变,导致LDL内化活性降低。罕见的人类枯草溶菌素转化酶9(proprotein convertase subtilisin/kexin type 9,PCSK9)发生功能型突变也可引起严重的FH表型。PCSK9通过降解LDLR蛋白间接下调LDL受体途径,其失功能突变可致血浆LDL水平下降。因此PCSK9是目前降脂药物的研究热点。
