WD-40 repeat protein SG2NA has multiple splice variants with tissue restricted and growth responsive properties

SG2NA is a member of the striatin family of WD-40 repeat proteins with potential scaffolding functions. It was originally identified as a tumor antigen with increased expression during S to G2 phase of cell cycle. We report here that mouse SG2NA has at least five novel splice variants of which two are devoid of the carboxyl terminal WD-40 repeats. The variants of SG2NA are generated by alternative splicing at the exon 7-9 regions and differ in their expression profiles in various tissues tested. While the 83, 78, 38 and 35 kDa variants are present in both brain and heart, the 87 kDa form is brain specific. Also, the expression of 35 kDa variant is more in neonatal than in adult tissues. Western analysis suggests that the SG2NA isoforms differentially respond to growth stimuli. Upon serum stimulation, while the 35 kDa variant is increased, the 78 kDa form is diminished. Splicing variation of SG2NA is conserved in metazoan evolution. In embryonic chicken there are at least four variants of which one is present in brain but absent in heart. Taken together, splicing variation of SG2NA might have some critical roles in differentiation and maturation in metazoan cells.     

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.     

High glucose upregulation of early-onset Parkinson's disease protein DJ-1 integrates the PRAS40/TORC1 axis to mesangial cell hypertrophy

The Akt kinase signaling pathway is frequently deregulated in many human diseases including cancer, autoimmune disease and diabetes. In nephropathy, associated with diabetes, increased Akt signal transduction results in glomerular especially mesangial cell hypertrophy. The mechanism of Akt activation by elevated glucose is poorly understood. The oncogene DJ-1 prevents oxidative damage and apoptosis of dopaminergic neurons in animal models of Parkinson's disease and in culture. We identified DJ-1 to increase in response to high glucose in renal glomerular mesangial cells concomitant with an increase in phosphorylation of Akt in a time-dependent manner. Plasmid-derived overexpression as well as downregulation of DJ-1 by siRNA showed the requirement of this protein in high glucose-stimulated Akt phosphorylation. The tumor suppressor protein PTEN acts as a negative regulator of Akt activation. Interestingly, DJ-1 was associated with PTEN and this interaction was significantly increased in response to high glucose. High glucose-induced increase in DJ-1 promoted phosphorylation of the PRAS40, a negative regulator of TORC1 kinase activity, resulting in activating and inactivating phosphorylation of S6 kinase and 4EBP-1, respectively. Furthermore, DJ-1 increased protein synthesis and hypertrophy of mesangial cells. Our results provide evidence for a unique mechanism whereby DJ-1 induces Akt/PRAS40/TORC1-mediated hypertrophy in response to high glucose.     

Reactive Oxygen Species-Mediated DJ-1 Monomerization Modulates Intracellular Trafficking Involving Karyopherin β2

Mutations in DJ-1 are a cause of recessive, early-onset Parkinson''s disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.     

DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein

DJ-1 is a ubiquitously expressed protein involved in various cellular processes including cell proliferation, RNA-binding, and oxidative stress. Mutations that result in loss of DJ-1 function lead to early onset parkinsonism in humans, and DJ-1 protein is present in pathological lesions of several tauopathies and synucleinopathies. In order to further investigate the role of DJ-1 in human neurodegenerative disease, we have generated novel polyclonal and monoclonal antibodies to human DJ-1 protein. We have characterized these antibodies and confirmed the pathological co-localization of DJ-1 with other neurodegenerative disease-associated proteins, as well as the decrease in DJ-1 solubility in disease tissue. In addition, we report the presence of DJ-1 in a large molecular complex (> 2000 kDa), and provide evidence for an interaction between endogenous DJ-1 and alpha-synuclein in normal and diseased tissue. These findings provide new avenues towards the study of DJ-1 function and how loss of its activity may lead to parkinsonism. Furthermore, our results provide further evidence for the interplay between neurodegenerative disease-associated proteins.     

人DJ-1蛋白在NIH3T3细胞中的表达与定位

采用增强型绿色荧光蛋白（EGFP）示踪的方法,研究人DJ-1蛋白在真核细胞中的 定位及其对刺激的反应. 将克隆在pGEX-KG上的DJ-1亚克隆到载体pEGFP-C2上,对 阳性克隆进行PCR、酶切和测序鉴定,用脂质体转染NIH3T3细胞;并用荧光显微镜观察 pEGFP-DJ-1在细胞内的定位以及在血清刺激时的移位;探讨在氧化应激时DJ-1对细胞的保护作用,以及其在细胞内定位的变化. 重组质粒在NIH3T3细胞中得到高效表达,绿色荧光弥散分布于胞质、胞核中,但以胞质居多;血清刺激后,细胞中的绿色荧光从胞质移位到胞核;在200～600 μmol/L H₂O₂刺激下,DJ-1能有效保护细胞抵抗氧化应激,并且也能从胞质移位到胞核.上述研究结果为进一步研究DJ-1蛋白的功能提供了一个重要依据.     

Zinedin, SG2NA, and striatin are calmodulin-binding, WD repeat proteins principally expressed in the brain

Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.     

Conservation of oxidative protein stabilization in an insect homologue of parkinsonism-associated protein DJ-1

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1α and DJ-1β) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1β. The structure of D. melanogaster DJ-1β is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1β. His126 in human DJ-1 is substituted with a tyrosine in DJ-1β, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO(2)(-)) results in considerable thermal stabilization of both Drosophila DJ-1β and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.     

The reversible low-temperature instability of human DJ-1 oxidative states

DJ-1 is a homodimeric protein that is centrally involved in various human diseases including Parkinson disease (PD). DJ-1 protects against oxidative damage and mitochondrial dysfunction through a homeostatic control of reactive oxygen species (ROS). DJ-1 pathology results from a loss of function, where ROS readily oxidizes a highly conserved and functionally essential cysteine (C106). The over-oxidation of DJ-1 C106 leads to a dynamically destabilized and biologically inactivated protein. An analysis of the structural stability of DJ-1 as a function of oxidative state and temperature may provide further insights into the role the protein plays in PD progression. NMR spectroscopy, circular dichroism, analytical ultracentrifugation sedimentation equilibrium, and molecular dynamics simulations were utilized to investigate the structure and dynamics of the reduced, oxidized (C106-SO₂⁻), and over-oxidized (C106-SO₃⁻) forms of DJ-1 for temperatures ranging from 5°C to 37°C. The three oxidative states of DJ-1 exhibited distinct temperature-dependent structural changes. A cold-induced aggregation occurred for the three DJ-1 oxidative states by 5°C, where the over-oxidized state aggregated at significantly higher temperatures than both the oxidized and reduced forms. Only the oxidized and over-oxidized forms of DJ-1 exhibited a mix state containing both folded and partially denatured protein that likely preserved secondary structure content. The relative amount of this denatured form of DJ-1 increased as the temperature was lowered, consistent with a cold-denaturation. Notably, the cold-induced aggregation and denaturation for the DJ-1 oxidative states were completely reversible. The dramatic changes in the structural stability of DJ-1 as a function of oxidative state and temperature are relevant to its role in PD and its functional response to oxidative stress.     

Drosophila DJ-1 mutants are selectively sensitive to environmental toxins associated with Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disorder that displays both sporadic and inherited forms. Exposure to several common environmental toxins acting through oxidative stress has been shown to be associated with PD. One recently identified inherited PD gene, DJ-1, may have a role in protection from oxidative stress, thus potentially linking a genetic cause with critical environmental risk factors. To develop an animal model that would allow integrative study of genetic and environmental influences, we have generated Drosophila lacking DJ-1 function. Fly DJ-1 homologs exhibit differential expression: DJ-1beta is ubiquitous, while DJ-1alpha is predominantly expressed in the male germline. DJ-1alpha and DJ-1beta double knockout flies are viable, fertile, and have a normal lifespan; however, they display a striking selective sensitivity to those environmental agents, including paraquat and rotenone, linked to PD in humans. This sensitivity results primarily from loss of DJ-1beta protein, which also becomes modified upon oxidative stress. These studies demonstrate that fly DJ-1 activity is selectively involved in protection from environmental oxidative insult in vivo and that the DJ-1beta protein is biochemically responsive to oxidative stress. Study of these flies will provide insight into the critical interplay of genetics and environment in PD.     

Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress

DJ-1 is a novel oncogene and causative gene for familial form of the Parkinson's disease (PD). DJ-1 has been shown to play a role in anti-oxidative stress by eliminating reactive oxygen species (ROS). The onset of PD is thought to be caused by oxidative stress and mitochondrial injury, which leads to protein aggregation that results in neuronal cell death. However, the mechanism by which DJ-1 triggers the onset of PD is still not clear. In this study, we analyzed association and localization of DJ-1 and its mutants with various chaperones. The results showed that DJ-1 and its mutants were associated with Hsp70, CHIP and mtHsp70/Grp75, a mitochondria-resident Hsp70, and that L166P and M26I mutants found in PD patients were strongly associated with Hsp70 and CHIP compared to wild-type and other DJ-1 mutants. DJ-1 and its mutants were colocalized with Hsp70 and CHIP in cells. Furthermore, association and colocalization of wildtype DJ-1 with mtHsp70 in mitochondria were found to be enhanced by treatment of cells with H₂O₂. These results suggest that translocation of DJ-1 to mitochondria after oxidative stress is carried out in association with chaperones.     

DJ-1 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson''s disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP⁺ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.     

Differential control of apoptosis by DJ-1 in prostate benign and cancer cells

DJ-1 is a conserved protein reported to be involved in diverse cellular processes ranging from cellular transformation, control of protein-RNA interaction, oxidative stress response to control of male infertility, among several others. Mutations in the human gene have been shown to be associated with an autosomal recessive, early onset Parkinson's disease (PARK7). The present study examines the control of DJ-1 expression in prostatic benign hyperplasia (BPH-1) and cancer (PC-3) cell lines in which DJ-1 abundance differs significantly. We show that while BPH-1 cells exhibit low basal level of DJ-1 expression, stress-inducing agents such as H(2)O(2) and mitomycin C markedly increase the intracellular level of the polypeptide. In contrast, DJ-1 expression is relatively high in PC-3 cells, and incubation with the same cytotoxic drugs does not modulate further the level of the polypeptide. In correlation with the expression of DJ-1, both cytotoxic agents activate the apoptotic pathway in the prostatic benign cells but not in PC-3 cells, which are resistant to their action. We further demonstrate that incubation of BPH-1 cells with TNF-related-apoptosis-inducing-ligand/Apo-2L (TRAIL) also enhances DJ-1 expression and that TRAIL and H(2)O(2) act additively to stimulate DJ-1 accumulation but synergistically in the activation of the apoptotic pathway. Time-course analysis of DJ-1 stimulation shows that while DJ-1 level increases without significant lag in TRAIL-treated cells, there is a delay in H(2)O(2)-treated cells, and that the increase in DJ-1 abundance precedes the activation of apoptosis. Unexpectedly, over-expression of DJ-1 de-sensitizes BPH-1 cells to the action of apoptotic-inducing agents. However, RNA-interference-mediated silencing of DJ-1 expression results in sensitization of PC-3 cells to TRAIL action. These results are consistent with a model in which DJ-1 is involved in the control of cell death in prostate cell lines. DJ-1 appears to play a differential role between cells expressing a low but inducible level of DJ-1 (e.g., BPH-1 cells) and those expressing a high but constitutive level of the polypeptide (e.g., PC-3 cells).     

Protection of Pancreatic β-Cells from Various Stress Conditions Is Mediated by DJ-1

Pancreatic β-cells are vulnerable to multiple stresses, leading to dysfunction and apoptotic death. Deterioration in β-cells function and mass is associated with type 2 diabetes. Comparative two-dimensional gel electrophoresis from pancreatic MIN6 cells that were maintained at varying glucose concentrations was carried out. An induced expression of a protein spot, detected in MIN6 cells experiencing high glucose concentration, was identified by mass spectrometry as the oxidized form of DJ-1. DJ-1 (park7) is a multifunctional protein implicated in familial Parkinsonism and neuroprotection in response to oxidative damage. The DJ-1 protein and its oxidized form were also induced following exposure to oxidative and endoplasmic reticulum stress in MIN6 and βTC-6 cells and also in mouse pancreatic islets. Suppression of DJ-1 levels by small interfering RNA led to an accelerated cell death, whereas an increase in DJ-1 levels by adenovirus-based infection attenuated cell death induced by H₂O₂ and thapsigargin in β-cell lines and mouse pancreatic islets. Furthermore, DJ-1 improved regulated insulin secretion under basal as well as oxidative and endoplasmic reticulum stress conditions in a dose-dependent manner. We identified TFII-I (Gtf2i) as DJ-1 partner in the cytosol, whereas the binding of TFII-I to DJ-1 prevented TFII-I translocation to the nucleus. The outcome was attenuation of the stress response. Our results suggest that DJ-1 together with TFII-I operate in concert to cope with various insults and to sustain pancreatic β-cell function.     

DISC1 promotes translation maintenance during sodium arsenite-induced oxidative stress

Variation in Disrupted-in-Schizophrenia 1 (DISC1) increases the risk for neurodegenerative diseases, schizophrenia, and other mental disorders. However, the functions of DISC1 associated with the development of these diseases remain unclear. DISC1 has been reported to inhibit Akt/mTORC1 signaling, a major regulator of translation, and recent studies indicate that DISC1 could exert a direct role in translational regulation. Here, we present evidence of a novel role of DISC1 in the maintenance of protein synthesis during oxidative stress. In order to investigate DISC1 function independently of Akt/mTORC1, we used Tsc2−/− cells, where mTORC1 activation is independent of Akt. DISC1 knockdown enhanced inhibition of protein synthesis in cells treated with sodium arsenite (SA), an oxidative agent used for studying stress granules (SGs) dynamics and translational control. N-acetyl-cysteine inhibited the effect of DISC1, suggesting that DISC1 affects translation in response to oxidative stress. DISC1 decreased SGs number in SA-treated cells, but resided outside SGs and maintained protein synthesis independently of a proper SG nucleation. DISC1-dependent stimulation of translation in SA-treated cells was supported by its interaction with eIF3h, a component of the canonical translation initiation machinery. Consistent with a role in the homeostatic maintenance of translation, DISC1 knockdown or overexpression decreased cell viability after SA exposure. Our data suggest that DISC1 is a relevant component of the cellular response to stress, maintaining certain levels of translation and preserving cell integrity. This novel function of DISC1 might be involved in its association with pathologies affecting tissues frequently exposed to oxidative stress.     

Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex–mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)–like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.