Nali Jia

DJ-1 Decreases Bax Expression through Repressing p53 Transcriptional Activity

DJ-1, originally identified as an oncogene product, is a protein with various functions in cellular transformation, oxidative stress response, and transcriptional regulation. Although previous studies suggest that DJ-1 is cytoprotective, the mechanism by which DJ-1 exerts its survival functions remains largely unknown. Here we show that DJ-1 exerts its cytoprotection through inhibiting p53-Bax-caspase pathway. DJ-1 interacts with p53 in vitro and in vivo. Overexpression of DJ-1 decreases the expression of Bax and inhibits caspase activation, whereas knockdown of DJ-1 increases Bax protein levels and accelerates caspase-3 activation and cell death induced by UV exposure. Our data provide evidence that the protective effects of DJ-1 on apoptosis are associated with its ability of decreasing Bax level through inhibiting p53 transcriptional activity.

Assembly of Lysine 63-linked Ubiquitin Conjugates by Phosphorylated α-Synuclein Implies Lewy Body Biogenesis

α-Synuclein (α-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of α-syn in LBs is phosphorylated at serine 129 (Ser129). However, the role of Ser129-phosphorylated α-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)α-syn and Ub, coexpression of phospho-mimic mutant α-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D α-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT α-syn, S129D α-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H2O2 and serum deprivation. These results suggest that the contribution of Ser129-phosphorylated α-syn to the Lys63-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.

Sumoylation is critical for DJ-1 to repress p53 transcriptional activity

Sumoylation is an important post‐translational modification, which is also involved in the pathogenesis of many neurodegenerative diseases. We previously reported that DJ‐1 decreases Bcl‐2 associated X protein expression through repressing p53 transcriptional activity. Here we show that DJ‐1(K130R), the non‐sumoylatable mutant form of DJ‐1, shifts from nucleus to cytoplasm, fails to repress p53 transcriptional activity and loses its protective function against ultraviolet induced cell death. Our findings suggest that sumoylation is critical for DJ‐1 to repress p53 transcriptional activity.

Nurr1 is phosphorylated by ERK2 in vitro and its phosphorylation upregulates tyrosine hydroxylase expression in SH-SY5Y cells.

Nurr1 is an orphan nuclear receptor essential for development and survival of dopaminergic neurons. Mutations in Nurr1 are associated with Parkinsons disease (PD) and there is a correlation between Nurr1 and tyrosine hydroxylase (TH) expression in PD brain. Two domains, activation function 1 (AF1) at the N-terminus and AF2 at the C-terminus of Nurr1, are important for Nurr1 activation. AF1 domain is conserved in NGFI-B/Nurr1/Nor-1 family members and MAPK signal pathway is involved in AF1 activity. Using in vitro phoshorylation assays, we have shown that ERK2 is a kinase to phosphorylate Nurr1 on multiple sites. S126 and T132, which are located near AF1 core of Nurr1, are dominant sites phosphorylated by ERK2. Moreover, using GST pull-down and co-IP assays, we identified that both the N-terminus of Nurr1 containing three ERK docking domains and another ERK docking domain in Nurr1 DNA binding domain are able to bind to ERK2. Furthermore, overexpression of a constitutively active form of MEK1, together with Nurr1 and mouse ERK2, greatly increases the tyrosine hydroxylase expression in SH-SY5Y cells. Reporter gene assays show that Nurr1Delta124-133/T185A, an ERK2 phospho-site mutant form, could not further increase its transcriptional activity on TH promoter, suggesting that Nurr1 phosphorylation by ERK2 may regulate its transcriptional activity on TH promoter. Thus, our results indicate that Nurr1 phosphorylation by ERK2 may play a role in regulating the TH expression.

p45, an ATPase subunit of the 19S proteasome, targets the polyglutamine disease protein ataxin-3 to the proteasome.

Machado–Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine tract near the C‐terminus of the MJD‐1 gene product, ataxin‐3. Ataxin‐3 is degraded by the proteasome. However, the precise mechanism of ataxin‐3 degradation remains to be elucidated. In this study, we show direct links between ataxin‐3 and the proteasome. p45, an ATPase subunit of the 19S proteasome, interacts with ataxin‐3 in vitro and stimulates the degradation of ataxin‐3 in an in vitro reconstituted degradation assay system. The effect of p45 on ataxin‐3 degradation is blocked by MG132, a proteasome inhibitor. In N2a or 293 cells, overexpression of p45 strikingly enhances the clearance of both normal and expanded ataxin‐3, but not alpha synuclein or SOD1, implying a functional specificity of p45 in this proteolytic process. The N‐terminus of ataxin‐3, which serves as a recognition site by p45, is necessary for the proteolytic process of ataxin‐3. We also show that other three ATPases of the 19S proteasome, MSS1, p48, and p56 have no effect on ataxin‐3 degradation. These data provide evidence that p45 plays an important role in regulating ataxin‐3 degradation by the proteasome.

PolyQ-expanded ataxin-3 interacts with full-length ataxin-3 in a polyQ length-dependent manner

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) tract in MJD-1 gene product, ataxin-3 (AT3). This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type (WT) AT3 and polyQ expanded AT3. Mouse neuroblastoma (N2a) cells or HEK293 cells were co-transfected with WT AT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays. Using fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner. WT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD. 脊髓小脑共济失调III型(spinocerebellar ataxia type 3, SCA3)/马查多-约瑟夫病(Machado-Joseph disease, MJD)是MJD-1基因中编码谷氨酰胺的密码子CAG的数量非正常扩增引起的一种多聚谷氨酰胺疾病, 也是神经退行性疾病的一种。 该病的主要病理特征为突变的ataxin-3在患者易感脑区的神经元胞核内聚集形成核内包涵体, 但其致病机制和突变蛋白在核内聚集的机制仍不清楚。 本研究是为了探讨突变型ataxin-3的病理学特性以及其聚集特性。 将野生型ataxin-3及不同多聚谷氨酰胺长度的突变型ataxin-3片段共转染到人胚胎肾细胞(HEK293 cells)和鼠成神经母细胞(neuroblastoma cells)中, 用荧光显微镜观察。 共转染48小时后, 收集在细胞中表达的蛋白, 用免疫印迹、 免疫共沉淀和GST-pull down实验检测蛋白的表达及结合。 用荧光显微镜观察到突变型ataxin-3片段在细胞内形成聚集, 其聚集体上募集有野生型全长ataxin-3; 突变型和野生型ataxin-3在细胞内聚集体上有共定位现象。 野生型ataxin-3片段并不形成聚集体, 与野生型全长ataxin-3在细胞内无聚集现象发生。 免疫共沉淀技术及GST-pulldown实验显示, 突变型ataxin-3片段与野生型全长ataxin-3存在相互作用, 且该作用强度呈现出多聚谷氨酰胺长度依赖性。 结果提示, 野生型ataxin-3通过与突变型ataxin-3片段的相互作用沉积于突变片段形成的聚集体中, 这可能影响野生型ataxin-3的正常功能, 进而可能对脊髓小脑共济失调III型(SCA3)/马查多-约瑟夫病(MJD)的发病产生影响。ObjectiveMachado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) tract in MJD-1 gene product, ataxin-3 (AT3). This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type (WT) AT3 and polyQ expanded AT3.MethodsMouse neuroblastoma (N2a) cells or HEK293 cells were co-transfected with WT AT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays.ResultsUsing fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner.ConclusionWT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD.摘要目的脊髓小脑共济失调III型(spinocerebellar ataxia type 3, SCA3)/马查多-约瑟夫病(Machado-Joseph disease, MJD)是MJD-1基因中编码谷氨酰胺的密码子CAG的数量非正常扩增引起的一种多聚谷氨酰胺疾病, 也是神经退行性疾病的一种。 该病的主要病理特征为突变的ataxin-3在患者易感脑区的神经元胞核内聚集形成核内包涵体, 但其致病机制和突变蛋白在核内聚集的机制仍不清楚。 本研究是为了探讨突变型ataxin-3的病理学特性以及其聚集特性。方法将野生型ataxin-3及不同多聚谷氨酰胺长度的突变型ataxin-3片段共转染到人胚胎肾细胞(HEK293 cells)和鼠成神经母细胞(neuroblastoma cells)中, 用荧光显微镜观察。 共转染48小时后, 收集在细胞中表达的蛋白, 用免疫印迹、 免疫共沉淀和GST-pull down实验检测蛋白的表达及结合。结果用荧光显微镜观察到突变型ataxin-3片段在细胞内形成聚集, 其聚集体上募集有野生型全长ataxin-3; 突变型和野生型ataxin-3在细胞内聚集体上有共定位现象。 野生型ataxin-3片段并不形成聚集体, 与野生型全长ataxin-3在细胞内无聚集现象发生。 免疫共沉淀技术及GST-pulldown实验显示, 突变型ataxin-3片段与野生型全长ataxin-3存在相互作用, 且该作用强度呈现出多聚谷氨酰胺长度依赖性。结论结果提示, 野生型ataxin-3通过与突变型ataxin-3片段的相互作用沉积于突变片段形成的聚集体中, 这可能影响野生型ataxin-3的正常功能, 进而可能对脊髓小脑共济失调III型(SCA3)/马查多-约瑟夫病(MJD)的发病产生影响。

PolyQ-expanded ataxin-3 interacts with full-length ataxin-3 in a polyQ length-dependent manner@@@多聚谷氨酰胺长度依赖性的突变型ataxin-3片段募集野生型ataxin-3

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) tract in MJD-1 gene product, ataxin-3 (AT3). This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type (WT) AT3 and polyQ expanded AT3. Mouse neuroblastoma (N2a) cells or HEK293 cells were co-transfected with WT AT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays. Using fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner. WT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD. 脊髓小脑共济失调III型(spinocerebellar ataxia type 3, SCA3)/马查多-约瑟夫病(Machado-Joseph disease, MJD)是MJD-1基因中编码谷氨酰胺的密码子CAG的数量非正常扩增引起的一种多聚谷氨酰胺疾病, 也是神经退行性疾病的一种。 该病的主要病理特征为突变的ataxin-3在患者易感脑区的神经元胞核内聚集形成核内包涵体, 但其致病机制和突变蛋白在核内聚集的机制仍不清楚。 本研究是为了探讨突变型ataxin-3的病理学特性以及其聚集特性。 将野生型ataxin-3及不同多聚谷氨酰胺长度的突变型ataxin-3片段共转染到人胚胎肾细胞(HEK293 cells)和鼠成神经母细胞(neuroblastoma cells)中, 用荧光显微镜观察。 共转染48小时后, 收集在细胞中表达的蛋白, 用免疫印迹、 免疫共沉淀和GST-pull down实验检测蛋白的表达及结合。 用荧光显微镜观察到突变型ataxin-3片段在细胞内形成聚集, 其聚集体上募集有野生型全长ataxin-3; 突变型和野生型ataxin-3在细胞内聚集体上有共定位现象。 野生型ataxin-3片段并不形成聚集体, 与野生型全长ataxin-3在细胞内无聚集现象发生。 免疫共沉淀技术及GST-pulldown实验显示, 突变型ataxin-3片段与野生型全长ataxin-3存在相互作用, 且该作用强度呈现出多聚谷氨酰胺长度依赖性。 结果提示, 野生型ataxin-3通过与突变型ataxin-3片段的相互作用沉积于突变片段形成的聚集体中, 这可能影响野生型ataxin-3的正常功能, 进而可能对脊髓小脑共济失调III型(SCA3)/马查多-约瑟夫病(MJD)的发病产生影响。ObjectiveMachado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) tract in MJD-1 gene product, ataxin-3 (AT3). This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type (WT) AT3 and polyQ expanded AT3.MethodsMouse neuroblastoma (N2a) cells or HEK293 cells were co-transfected with WT AT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays.ResultsUsing fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner.ConclusionWT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD.摘要目的脊髓小脑共济失调III型(spinocerebellar ataxia type 3, SCA3)/马查多-约瑟夫病(Machado-Joseph disease, MJD)是MJD-1基因中编码谷氨酰胺的密码子CAG的数量非正常扩增引起的一种多聚谷氨酰胺疾病, 也是神经退行性疾病的一种。 该病的主要病理特征为突变的ataxin-3在患者易感脑区的神经元胞核内聚集形成核内包涵体, 但其致病机制和突变蛋白在核内聚集的机制仍不清楚。 本研究是为了探讨突变型ataxin-3的病理学特性以及其聚集特性。方法将野生型ataxin-3及不同多聚谷氨酰胺长度的突变型ataxin-3片段共转染到人胚胎肾细胞(HEK293 cells)和鼠成神经母细胞(neuroblastoma cells)中, 用荧光显微镜观察。 共转染48小时后, 收集在细胞中表达的蛋白, 用免疫印迹、 免疫共沉淀和GST-pull down实验检测蛋白的表达及结合。结果用荧光显微镜观察到突变型ataxin-3片段在细胞内形成聚集, 其聚集体上募集有野生型全长ataxin-3; 突变型和野生型ataxin-3在细胞内聚集体上有共定位现象。 野生型ataxin-3片段并不形成聚集体, 与野生型全长ataxin-3在细胞内无聚集现象发生。 免疫共沉淀技术及GST-pulldown实验显示, 突变型ataxin-3片段与野生型全长ataxin-3存在相互作用, 且该作用强度呈现出多聚谷氨酰胺长度依赖性。结论结果提示, 野生型ataxin-3通过与突变型ataxin-3片段的相互作用沉积于突变片段形成的聚集体中, 这可能影响野生型ataxin-3的正常功能, 进而可能对脊髓小脑共济失调III型(SCA3)/马查多-约瑟夫病(MJD)的发病产生影响。

SUMO-1 modification increases human SOD1 stability and aggregation

The mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause approximately 20% cases of familial amyotrophic lateral sclerosis (FALS), characterized by selective loss of motor neurons. Mutant SOD1 forms inclusions in tissues from FALS patients. However, the precise mechanism of the accumulation of mutant SOD1 remains unclear. Here we show that human SOD1 is a substrate modified by SUMO-1. A conversion of lysine 75 to an arginine within a SUMO consensus sequence in SOD1 completely abolishes SOD1 sumoylation. We further show that SUMO-1 modification, on both wild-type and mutant SOD1, increases SOD1 steady state level and aggregation. Moreover, SUMO-1 co-localizes onto the aggregates formed by SOD1. These findings imply that SUMO-1 modification on lysine 75 may participate in regulating SOD1 stability and its aggregation process. Thus, our results suggest that sumoylation of SOD1 may be involved in the pathogenesis of FALS associated with mutant SOD1.