Erkang Fei

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.

Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation

Superoxide dismutase-1 (SOD1) and ataxin-3 are two neurodegenerative disease proteins in association with familial amyotrophic lateral sclerosis and Machado-Joseph disease/spinocerebellar ataxia type 3. Both normal and mutant types of SOD1 and ataxin-3 are degraded by the proteasome. It was recently reported that these two proteins are associated with the endoplasmic reticulum (ER). Mammalian gp78 is an E3 ubiquitin ligase involved in ER-associated degradation (ERAD). Here, we show that gp78 interacts with both SOD1 and ataxin-3. Overexpression of gp78 promotes the ubiquitination and degradation of these two proteins, whereas knockdown of gp78 stabilizes them. Moreover, gp78 represses aggregate formation of mutant SOD1 and protect cells against mutant SOD1-induced cell death. Furthermore, gp78 is increased in cells transfected with these two mutant proteins as well as in ALS mice. Thus, our results suggest that gp78 functions in the regulation of SOD1 and ataxin-3 to target them for ERAD.

Omi/HtrA2 is a positive regulator of autophagy that facilitates the degradation of mutant proteins involved in neurodegenerative diseases

Omi, also known as high temperature requirement factor A2 (HtrA2), is a serine protease that was originally identified as a proapoptotic protein. Like Smac/Diablo, it antagonizes inhibitor of apoptosis proteins when released into the cytosol on apoptotic stimulation. Loss of its protease activity in mnd2 (motor neuron degeneration 2) mice is associated with neurodegeneration. However, the detailed mechanisms by which Omi regulates the pathogenesis of neurodegenerative disease remain largely unknown. We report here that Omi participates in the pivotal cellular degradation process known as autophagy. It activates autophagy through digestion of Hax-1, a Bcl-2 family-related protein that represses autophagy in a Beclin-1 (mammalian homologue of yeast ATG6)-dependent pathway. Moreover, Omi-induced autophagy facilitates the degradation of neurodegenerative proteins such as pathogenic A53T α-synuclein and truncated polyglutamine-expanded huntingtin, as well as the endogenous autophagy substrate p62. Knockdown of Omi decreases the basal level of autophagy and increases the level of the above target proteins. Furthermore, S276C Omi, the protease-defective mutant found in mnd2 mice, fails to regulate autophagy. Increased autophagy substrates and the formation of aggregate structures are observed in the brains of mnd2 mice. These results identify Omi as a novel regulator of autophagy and suggest that Omi might be important in the cellular quality control of proteins involved in neurodegenerative diseases.

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.

Dysbindin-1, a schizophrenia-related protein, facilitates neurite outgrowth by promoting the transcriptional activity of p53

Genetic variations in the DTNBP1 gene (encoding the protein dysbindin-1) have been implicated as risk factors in the pathogenesis of schizophrenia. Previous studies have indicated that dysbindin-1 functions in the regulation of synaptic activity. Recently, dysbindin-1 has also been documented to be involved in neuronal development. In this study, we identified necdin as a binding partner of dysbindin-1 using a yeast two-hybrid screen. Dysbindin-1 recruits necdin to the cytoplasm, thereby attenuating the repressive effects of necdin on p53 transcriptional activity. Knockdown of dysbindin-1, like knockdown of p53, greatly decreases the expressions of the p53 target genes coronin 1b and rab13, which are required for neurite outgrowth. Moreover, overexpression of p53 restores the neurite outgrowth blocked by dysbindin-1 knockdown. In brains of dysbindin-1 null mice (the sandy strain), p21, Coronin 1b and Rab13 levels are reduced. Furthermore, primary cultured cortical neurons from sandy mice display neurite outgrowth defects when compared with those from wild-type mice. Thus, our data provide evidence that dysbindin-1 has an important role in neurite outgrowth through its regulation of p53’s 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.

The KDEL receptor induces autophagy to promote the clearance of neurodegenerative disease-related proteins

Endoplasmic reticulum (ER) stress is involved in neurodegenerative diseases, and the KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR) plays a key role in ER quality control and in the ER stress response. The subcellular distribution of KDELR is dynamic and related to its ligand binding status and its expression level. Here, we show that KDELR mRNA is upregulated upon thapsigargin treatment, which induces ER stress. Moreover, overexpressed KDELR partially redistributes to the lysosome and activates autophagy. The R169N mutant, a ligand binding-defective form of KDELR, and D193N, a transport-defective form of KDELR, both fail to trigger autophagy. Overexpression of KDELR activates extracellular signal-regulated kinases (ERKs). Both the activation of ERKs and autophagy induced by KDELR could be blocked by PD98059, an inhibitor of mitogen extracellular kinase 1 (MEK1). The overexpression of some neurodegenerative disease-related proteins, such as amyotrophic lateral sclerosis (ALS)-linked G93A superoxide dismutase 1 (SOD1), Parkinsons disease-associated A53T alpha-synuclein and Huntingtons disease-related expanded huntingtin, increase the mRNA levels of KDELR. Moreover, the overexpressed KDELR promotes the clearance of these disease proteins through autophagy. Taken together, our data provide evidence that KDELR, as a novel inducer of autophagy, participates in the degradation of misfolded neurodegenerative disease-related proteins.

DJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8 recruitment to FADD

DJ-1 was initially identified as an oncogene product involved in human tumorigenesis in cooperation with Ras. Increased DJ-1 expression is associated with tumorigenesis in many cancers, whereas the loss of DJ-1 function is linked to an autosomal recessive form of Parkinsons disease (PD). It has been reported that DJ-1 protects cells from TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, the mechanism by which DJ-1 is involved is still largely unknown. Here we show that DJ-1 inhibits TRAIL-induced apoptosis by blocking Fas-associated protein death domain (FADD)-mediated pro-caspase-8 activation. Wild-type DJ-1, but not the PD-associated mutant L166P, binds to FADD to inhibit the formation of the death-inducing signaling complex (DISC). DJ-1 competes with pro-caspase-8 to bind to FADD at the death effector domain, thereby repressing the recruitment and activation of pro-caspase-8 to the active form of caspase-8. Thus, our study suggests that DJ-1 protects against TRAIL-induced apoptosis through the regulation of DISC formation.

Nucleocytoplasmic Shuttling of Dysbindin-1, a Schizophrenia-related Protein, Regulates Synapsin I Expression

Dysbindin-1 is a 50-kDa coiled-coil-containing protein encoded by the gene DTNBP1 (dystrobrevin-binding protein 1), a candidate genetic factor for schizophrenia. Genetic variations in this gene confer a susceptibility to schizophrenia through a decreased expression of dysbindin-1. It was reported that dysbindin-1 regulates the expression of presynaptic proteins and the release of neurotransmitters. However, the precise functions of dysbindin-1 are largely unknown. Here, we show that dysbindin-1 is a novel nucleocytoplasmic shuttling protein and translocated to the nucleus upon treatment with leptomycin B, an inhibitor of exportin-1/CRM1-mediated nuclear export. Dysbindin-1 harbors a functional nuclear export signal necessary for its nuclear export, and the nucleocytoplasmic shuttling of dysbindin-1 affects its regulation of synapsin I expression. In brains of sandy mice, a dysbindin-1-null strain that displays abnormal behaviors related to schizophrenia, the protein and mRNA levels of synapsin I are decreased. These findings demonstrate that the nucleocytoplasmic shuttling of dysbindin-1 regulates synapsin I expression and thus may be involved in the pathogenesis of schizophrenia.