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
AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heartspecific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.
oxidoreductase iron-sulfur protein 4; NSUN3, NOL1/NOP2/Sun domain-containing protein 3; OCR, oxygen consumption rate; OG, n-octyl-β-D-glucoside; OXPHOS, oxidative phosphorylation; RC, respiratory chain; ROS, reactive oxygen species. Correspondence AbstractThe respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP] mito ) in a zebrafish hypoxia model. Heartspecific Higd1a overexpression mitigated the decline in [ATP] mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease;notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal 1860 | NAGAO et Al.
33 34 Running title 35 AMPK regulates shape of cardiomyocytes 36 2 Summary 37AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule 38 (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological 39 relevance in vivo remains to be elucidated. In this study, we identified an active form of 40 AMPK localized at the intercalated discs in the heart, a specific cell-cell junction present 41 between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of 42 AMPK at the intercalated discs, and the effect was reversed by the removal of 43 suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse 44 imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK 45 leads to the accumulation of MTs at the intercalated discs. Interestingly, MYK-461 increased 46 the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. 47Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of 48 cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac 49 contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and 50 aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic 51 phosphorylation of CLIP-170 at the intercalated discs. 52 53 54 55 Keywords 56 AMPK, microtubule, CLIP-170, intercalated disc 57 58 59 60 61 62 Introduction 63AMP-activated protein kinase (AMPK) can sense the increase of intracellular AMP or ADP 64 concentration, and is fully activated by the phosphorylation of conserved Thr residue in the 65 activation loop by upstream kinases, including LKB1 or Ca 2+ /calmodulin-activated protein 66 kinase kinases, CaMKK2 [1]. Canonical stimulation known to activate AMPK is energetic 67 stress, and this explains why AMPK switches on downstream signaling pathways involved in 68 ATP production while switching off the anabolic pathways. However, AMPK can be activated 69 by various stimuli other than energetic stresses including Ca 2+ increase, oxidative stress, or 70 genotoxic stress. The downstream effects of AMPK are not just restricted to the regulation of 71 metabolism. It has also been demonstrated that AMPK is a multifunctional kinase known to 72 regulate cell cycle, polarity, membrane excitability, and a variety of cellular functions by 73 phosphorylating specific sets of substrates, presumably in a spatiotemporal manner [1][2][3]. 74As such, we previously demonstrated that AMPK controls directional cell migration by 75 modulating microtubule (MT) dynamic instability through direct phosphorylation at CLIP-170 76 Ser-311 in Vero cells [4]. MTs are noncovalent polymers comprised of tubulin heterodimers, 77 and one of the major constituents of cytoskeleton. Although the term of cytoskeleton 78 suggests static structure, MTs are in fact highly dynamic, especially the plus end of MTs, 79 which exhibits a behavior called as dynamic instability; individua...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.