“…To evaluate whether PARK7 secretion was mediated by the conventional ER-/Golgi-dependent secretion mechanism, cells were treated with brefeldin A, an inhibitor of ER-Golgi transport, as a result of which it was found that treatment with brefeldin A inhibited FN1 secretion but not PARK7 secretion (Figure 1(C), suggesting that the conventional secretory pathway was not involved in PARK7 secretion. As previously reported [9,10], most of PARK7 was found to be in the cytosolic protein-enriched fraction obtained by subcellular fractionation (Figure 1(D)), supporting the idea that PARK7 was secreted via an ER-/Golgi-independent secretory pathway. We also used 2D-PAGE to examine the oxidative state of PARK7, as a result of which we found that the ratio of oxPARK7 to total PARK7 in medium was almost the same as that in cells, suggesting that secretion of PARK7 was not induced by its oxidation (Figure 1(E)).10.1080/15548627.2018.1493043-F0001Figure 1.PARK7 was secreted from SH-SY5Y cells.…”
Section: Resultssupporting
confidence: 90%
“…PARK7/DJ-1 (Parkinsonism associated deglycase) was initially identified as an oncoprotein that cooperates with Ras to promote cell transformation [9]. PARK7 has been implicated as a protein encoded by one of the causative genes ( PARK7 ) in a familial form of Parkinson disease (PD) [10].…”
Section: Introductionmentioning
confidence: 99%
“…PARK7 is localized in the cytoplasm and the nucleus; it has also been shown that some PARK7 is localized in the mitochondria [9,10,19,20]. Several lines of evidence suggest that PARK7 is secreted from cells into the circulation despite the fact that PARK7 lacks a secretory signal sequence [21–29].…”
PARK7/DJ-1 is a Parkinson disease- and cancer-associated protein that functions as a multifunctional protein involved in gene transcription regulation and anti-oxidative defense. Although PARK7 lacks the secretory signal sequence, it is secreted and plays important physiological and pathophysiological roles. Whereas secretory proteins that lack the endoplasmic reticulum-targeting signal sequence are secreted from cells by way of what is called the unconventional secretion mechanism, the specific processes responsible for causing PARK7 to be secreted across the plasma membrane have remained unclear. In the present study, we found that PARK7 secretion was increased by treatment with 6-OHDA via the unconventional secretory pathway in human neuroblastoma SH-SY5Y cells and MEF cells. We also found that 6-OHDA-induced PARK7 secretion was suppressed in Atg5-, Atg9-, or Atg16l1-deficient MEF cells or ATG16L1 knockdown SH-SY5Y cells, indicating that the autophagy-based unconventional secretory pathway is involved in PARK7 secretion. We moreover observed that 6-OHDA-derived electrophilic quinone induced oxidative stress as indicated by a decrease in glutathione levels, and that this was suppressed by pretreatment with antioxidant NAC. We further found that NAC treatment suppressed autophagy and PARK7 secretion. We also observed that 6-OHDA-induced autophagy was associated with activation of AMPK and ULK1 via a pathway which was independent of MTOR. Collectively these results suggest that electrophilic 6-OHDA quinone enhances oxidative stress, and that this is followed by AMPK-ULK1 pathway activation and induction of secretory autophagy to produce unconventional secretion of PARK7.Abbreviations: 6-OHDA: 6-hydroxydopamine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CAV1: caveolin 1; ER: endoplasmic reticulum; FN1: fibronectin 1; GSH: glutathione; IDE: insulin degrading enzyme; IL: interleukin; LDH: lactate dehydrogenase; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PARK7/DJ-1: Parkinsonism associated deglycase; PD: Parkinson disease; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; RPN1: ribophorin I; ROS: reactive oxygen species; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type
“…To evaluate whether PARK7 secretion was mediated by the conventional ER-/Golgi-dependent secretion mechanism, cells were treated with brefeldin A, an inhibitor of ER-Golgi transport, as a result of which it was found that treatment with brefeldin A inhibited FN1 secretion but not PARK7 secretion (Figure 1(C), suggesting that the conventional secretory pathway was not involved in PARK7 secretion. As previously reported [9,10], most of PARK7 was found to be in the cytosolic protein-enriched fraction obtained by subcellular fractionation (Figure 1(D)), supporting the idea that PARK7 was secreted via an ER-/Golgi-independent secretory pathway. We also used 2D-PAGE to examine the oxidative state of PARK7, as a result of which we found that the ratio of oxPARK7 to total PARK7 in medium was almost the same as that in cells, suggesting that secretion of PARK7 was not induced by its oxidation (Figure 1(E)).10.1080/15548627.2018.1493043-F0001Figure 1.PARK7 was secreted from SH-SY5Y cells.…”
Section: Resultssupporting
confidence: 90%
“…PARK7/DJ-1 (Parkinsonism associated deglycase) was initially identified as an oncoprotein that cooperates with Ras to promote cell transformation [9]. PARK7 has been implicated as a protein encoded by one of the causative genes ( PARK7 ) in a familial form of Parkinson disease (PD) [10].…”
Section: Introductionmentioning
confidence: 99%
“…PARK7 is localized in the cytoplasm and the nucleus; it has also been shown that some PARK7 is localized in the mitochondria [9,10,19,20]. Several lines of evidence suggest that PARK7 is secreted from cells into the circulation despite the fact that PARK7 lacks a secretory signal sequence [21–29].…”
PARK7/DJ-1 is a Parkinson disease- and cancer-associated protein that functions as a multifunctional protein involved in gene transcription regulation and anti-oxidative defense. Although PARK7 lacks the secretory signal sequence, it is secreted and plays important physiological and pathophysiological roles. Whereas secretory proteins that lack the endoplasmic reticulum-targeting signal sequence are secreted from cells by way of what is called the unconventional secretion mechanism, the specific processes responsible for causing PARK7 to be secreted across the plasma membrane have remained unclear. In the present study, we found that PARK7 secretion was increased by treatment with 6-OHDA via the unconventional secretory pathway in human neuroblastoma SH-SY5Y cells and MEF cells. We also found that 6-OHDA-induced PARK7 secretion was suppressed in Atg5-, Atg9-, or Atg16l1-deficient MEF cells or ATG16L1 knockdown SH-SY5Y cells, indicating that the autophagy-based unconventional secretory pathway is involved in PARK7 secretion. We moreover observed that 6-OHDA-derived electrophilic quinone induced oxidative stress as indicated by a decrease in glutathione levels, and that this was suppressed by pretreatment with antioxidant NAC. We further found that NAC treatment suppressed autophagy and PARK7 secretion. We also observed that 6-OHDA-induced autophagy was associated with activation of AMPK and ULK1 via a pathway which was independent of MTOR. Collectively these results suggest that electrophilic 6-OHDA quinone enhances oxidative stress, and that this is followed by AMPK-ULK1 pathway activation and induction of secretory autophagy to produce unconventional secretion of PARK7.Abbreviations: 6-OHDA: 6-hydroxydopamine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CAV1: caveolin 1; ER: endoplasmic reticulum; FN1: fibronectin 1; GSH: glutathione; IDE: insulin degrading enzyme; IL: interleukin; LDH: lactate dehydrogenase; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PARK7/DJ-1: Parkinsonism associated deglycase; PD: Parkinson disease; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; RPN1: ribophorin I; ROS: reactive oxygen species; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type
“…These factors lead to oxidative stress, mitochondrial dysfunction and impairment of the protein degradation system, resulting in cell death. DJ-1 was first identified by our group as a novel oncogene product [1] and was later found to be a causative gene product of a familial form of PD, PARK7 [2]. DJ-1 plays roles in transcriptional regulation [3][4][5][6][7][8] and anti-oxidative stress reaction [9][10][11][12], and loss of its function is thought to affect the onset of PD.…”
Section: Introductionmentioning
confidence: 99%
“…These findings indicate that the function of mitochondrial complex I is related to the onset of PD. DJ-1 is located both in the cytoplasm and nucleus [1] and has recently been shown to be located in mitochondria [11,[24][25][26]. It has also been reported that some parts of DJ-1 were translocated into mitochondria after cells had been subjected to oxidative stress to protect cells from oxidative stress-induced cell death [27][28][29][30].…”
Parkinson's disease (PD) is caused by neuronal cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for onset of PD. DJ-1, a causative gene product of a familial form of Parkinson's disease, PARK7, plays roles in transcriptional regulation and anti-oxidative stress. The possible mitochondrial function of DJ-1 has been proposed, but its exact function remains unclear. In this study, we found that DJ-1 directly bound to NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and was co-localized with complex I and that complex I activity was reduced in DJ-1-knockdown NIH3T3 and HEK293cells. These findings suggest that DJ-1 is an integral mitochondrial protein and that DJ-1 plays a role in maintenance of mitochondrial complex I activity.
In a previous study [Nachaliel et al., 1993], we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed.
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