In mammals, autophagosome formation is initiated by ULK1 via the posttranslational modification of this protein. However, the precise role of ULK1 ubiquitination in modulating autophagy is unknown. Here, we show that NEDD4L, an E3 ubiquitin ligase, binds ULK1 in pancreatic cancer cells. ULK1 expression was stabilized in NEDD4L knockdown cells compared to that in control cells, suggesting that NEDD4L is involved in ULK1 ubiquitination and its subsequent degradation. Autophagy activity was enhanced in NEDD4L knockdown cells compared to control cells. NEDD4L-depleted cells exhibited an increase in the cellular oxygen consumption rate (OCR) and mitochondrial membrane potential, and maintained mitochondrial fusion status in response to metabolic stress. Enhanced OCR and mitochondrial fusion morphology in NEDD4L knockdown cells were repressed by siRNA targeting ULK1. In addition to ULK1, ASCT2, a glutamine transporter, was accumulated in NEDD4L-depleted cells; this is important for maintaining autophagy activation and mitochondrial metabolic function. Finally, the cellular growth and survival rate increased in NEDD4L knockdown cells compared to control cells. However, the genetic or pharmacological blockade of either ULK1 or ASCT2 in NEDD4L-depleted cells sensitized pancreatic cancer cells, particularly in response to nutrient deprivation. In a mouse xenograft model of pancreatic cancer, the use of autophagy inhibitors suppressed tumor growth more in NEDD4L-depleted cells than in tumors from control cells. NEDD4L and ULK1 levels were inversely correlated in two different pancreatic cancer mouse models-xenograft mouse and KPC mouse models. These results suggest that NEDD4L suppressed autophagy and mitochondrial metabolism by reducing cellular ULK1 or ASCT2 levels, and thus could repress the growth and survival of pancreatic cancer cells. Therefore, ubiquitin ligase-mediated autophagy plays a critical role in regulating mitochondrial metabolism, thereby contributing to the growth and survival of certain cancers with low NEDD4L levels.
The initiation of macroautophagy/autophagy is tightly regulated by the upstream ULK1 kinase complex, which affects many downstream factors including the PtdIns3K complex. The phosphorylation of the right position at the right time on downstream molecules is governed by proper complex formation. One component of the ULK1 complex, ATG101, known as an accessory protein, is a stabilizer of ATG13 in cells. The WF finger region of ATG101 plays an important role in the recruitment of WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) and ZFYVE1 (zinc finger FYVE-type containing 1). Here, we report that the C-terminal region identified in the structure of the human ATG101-ATG13 complex is responsible for the binding of the PtdIns3K complex. This region adopts a β-strand conformation in free ATG101, but either an α-helix or random coil in our ATG101-ATG13 complex, which protrudes from the core and interacts with other molecules. The C-terminal deletion of ATG101 shows a significant defect in the interaction with PtdIns3K components and subsequently impairs autophagosome formation. This result clearly presents an additional role of ATG101 for bridging the ULK1 and PtdIns3K complexes in the mammalian autophagy process. Abbreviations: ATG: autophagy related; BECN1: beclin 1; GFP: green fluorescent protein; HORMA: Hop1p/Rev7p/MAD2; HsATG13: HORMA domain of ATG13 from Homo sapiens; KO: knockout; MAD2: mitotic arrest deficient 2 like 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SAXS: small-angle X-ray scattering; ScAtg13: HORMA domain of Atg13 from Sccharomyces cerevisiae; SEC-SAXS: size-exclusion chromatography with small-angle X-ray scattering; SpAtg13: HORMA domain of Atg13 from Schizosaccharomyces pombe; SQSTM1/p62: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain: phosphoinositide interacting 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.
Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis.
Recent years has seen research attention into the therapeutic value of Relational Depth (RD).RD refers to moments in a therapeutic relationship in which a person has feelings of aliveness, satisfaction and immersion. However, no research has yet tested for the association between RD and concepts closely aligned with Carl Rogers' hypothesis of how people change in a growth promoting relationship. In this study, 55 therapy clients completed the Relational Depth Inventory (RDI), the Unconditional Positive Self-Regard Scale (UPSR) and the Authenticity Scale (AS). It was found that higher scores on the RDI were associated with higher scores on the UPSR and the AS. These results provide initial evidence for the growth promoting effects of RD. Further prospective research is now warranted.
Background The World Health Organization announced the inclusion of gaming disorder (GD) in the International Classification of Diseases, 11th Revision, despite some concerns. However, video gaming has been associated with the enhancement of cognitive function. Moreover, despite comparable extensive video gaming, pro gamers have not shown any of the negative symptoms that individuals with GD have reported. It is important to understand the association between extensive video gaming and alterations in brain regions more objectively. Objective This study aimed to systematically explore the association between extensive video gaming and changes in cognitive function by focusing on pro gamers and individuals with GD. Methods Studies about pro gamers and individuals with GD were searched for in the PubMed and Web of Science databases using relevant search terms, for example, “pro-gamers” and “(Internet) gaming disorder.” While studies for pro gamers were searched for without date restrictions, only studies published since 2013 about individuals with GD were included in search results. Article selection was conducted by following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Results By following the PRISMA guidelines, 1903 records with unique titles were identified. Through the screening process of titles and abstracts, 86 full-text articles were accessed to determine their eligibility. A total of 18 studies were included in this systematic review. Among the included 18 studies, six studies included pro gamers as participants, one study included both pro gamers and individuals with GD, and 11 studies included individuals with GD. Pro gamers showed structural and functional alterations in brain regions (eg, the left cingulate cortex, the insula subregions, and the prefrontal regions). Cognitive function (eg, attention and sensorimotor function) and cognitive control improved in pro gamers. Individuals with GD showed structural and functional alterations in brain regions (eg, the striatum, the orbitofrontal cortex, and the amygdala) that were associated with impaired cognitive control and higher levels of craving video game playing. They also showed increased cortical thickness in the middle temporal cortex, which indicated the acquisition of better skills. Moreover, it was suggested that various factors (eg, gaming expertise, duration or severity of GD, and level of self-control) seemed to modulate the association of extensive video game playing with changes in cognitive function. Conclusions Although a limited number of studies were identified that included pro gamers and/or individuals who reported showing symptoms of GD for more than 1 year, this review contributed to the objective understanding of the association between extensive video game playing and changes in cognitive function. Conducting studies with a longitudinal design or with various comparison groups in the future would be helpful in deepening the understanding of this association.
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