Abstract 174: The BH3-Only Protein BNIP3 Induces Mitochondrial Clearance via Multiple Pathways

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Eileen R Gonzalez ◽  
Babette Hammerling ◽  
Rita Hanna ◽  
Dieter A Kubli ◽  
Åsa B Gustafsson
Keyword(s):  
Cell Death ◽  
Ubiquitin Ligase ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Wild Type ◽  
Potent Inducer ◽  
Autophagy Pathway ◽  
Endosomal Pathway ◽  
In Cells

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.

scholarly journals Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Myungjin Kim ◽  
Erin Sandford ◽  
Damian Gatica ◽  
Yu Qiu ◽  
Xu Liu ◽  
...  
Keyword(s):  
Developmental Delay ◽  
Neurodegenerative Disorders ◽  
Neurological Diseases ◽  
Critical Role ◽  
Wild Type ◽  
Autophagy Flux ◽  
The Core ◽  
Atg Genes ◽  
Autophagy Pathway

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

scholarly journals Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

2016 ◽  
Vol 113 (37) ◽  
pp. 10394-10399 ◽  
Author(s):  
Sinyi Kong ◽  
Yi Yang ◽  
Yuanming Xu ◽  
Yajun Wang ◽  
Yusi Zhang ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Death Receptor ◽  
Cell Immunity ◽  
B Cell Immunity

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.

scholarly journals E3 ubiquitin ligase SYVN1 is a key positive regulator for GSDMD-mediated pyroptosis

2021 ◽  
Author(s):  
Yuhua Shi ◽  
Yang Yang ◽  
Weilv Xu ◽  
Wei Xu ◽  
Xinyu Fu ◽  
...  
Keyword(s):  
Cell Death ◽  
Ubiquitin Ligase ◽  
Essential Role ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
E3 Ubiquitin Ligase ◽  
Positive Regulator ◽  
Ldh Release

Gasdermin D (GSDMD) participates in activation of inflammasomes and pyroptosis. Meanwhile, ubiquitination strictly regulates inflammatory responses. However, how ubiquitination regulates Gasdermin D activity is not well understood. In this study, we show that pyroptosis triggered by Gasdermin D is regulated through ubiquitination. Specifically, SYVN1, an E3 ubiquitin ligase of gasdermin D, promotes GSDMD-mediated pyroptosis. SYVN1 deficiency inhibits pyroptosis and subsequent LDH release and PI uptake. SYVN1 directly interacts with GSDMD, and mediates K27-linked polyubiquitination of GSDMD on K203 and K204 residues, promoting GSDMD-induced pyroptotic cell death. Thus, our findings revealed the essential role of SYVN1 in GSDMD-mediated pyroptosis. Overall, GSDMD ubiquitination is a potential therapeutic module for inflammatory diseases.

scholarly journals Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

2018 ◽  
Vol 72 (4) ◽  
pp. 739-752.e9 ◽  
Author(s):  
Benjamin M. Foster ◽  
Paul Stolz ◽  
Christopher B. Mulholland ◽  
Alex Montoya ◽  
Holger Kramer ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligase Activity

scholarly journals The E3 Ubiquitin Ligase ATL9 Affects Expression of Defense Related Genes, Cell Death and Callose Deposition in Response to Fungal Infection

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 68
Author(s):  
Tingwei Guo ◽  
Feng Kong ◽  
Carter Burton ◽  
Steven Scaglione ◽  
Blake Beagles ◽  
...  
Keyword(s):  
Cell Death ◽  
Fungal Infection ◽  
Ubiquitin Ligase ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Defense Responses ◽  
Callose Deposition ◽  
Expression Levels ◽  
Insertional Mutants ◽  
Protein Ubiquitination

Plants use diverse strategies to defend themselves from biotic stresses in nature, which include the activation of defense gene expression and a variety of signal transduction pathways. Previous studies have shown that protein ubiquitination plays a critical role in plant defense responses, however the details of its function remain unclear. Our previous work has shown that increasing expression levels of ATL9, an E3 ubiquitin ligase in Arabidopsis thaliana, increased resistance to infection by the fungal pathogen, Golovinomyces cichoracearum. In this study, we demonstrate that the defense-related proteins PDF1.2, PCC1 and FBS1 directly interact with ATL9 and are targeted for degradation to the proteasome by ATL9. The expression levels of PDF1.2, PCC1 and FBS1 are decreased in T-DNA insertional mutants of atl9 and T-DNA insertional mutants of pdf1.2, pcc1 and fbs1 are more susceptible to fungal infection. In addition, callose is more heavily deposited at infection sites in the mutants of atl9, fbs1, pcc1 and pdf1.2. Overexpression of ATL9 and of mutants in fbs1, pcc1 and pdf1.2 showed increased levels of cell death during infection. Together these results indicate that ubiquitination, cell death and callose deposition may work together to enhance defense responses to fungal pathogens.

scholarly journals Oncogenic Mechanisms of CBL E3 Ubiquitin Ligase Mutations in Myeloid Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 563-563
Author(s):  
Roger Belizaire ◽  
Sebastian Koochaki ◽  
Namrata Udeshi ◽  
Alexis Vedder ◽  
Lei Sun ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Research Funding ◽  
E3 Ubiquitin Ligase ◽  
Akt Signaling ◽  
Myeloid Malignancies ◽  
Proliferative Advantage ◽  
In Cells

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.

Abstract 297: Crataegus Special Extract WS 1442 Retards Replicative Senescence in Coronary Artery Endothelial Cells: Role of eNOS-derived NO

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Noureddine Idris-Khodja ◽  
Marouene Kheloufi ◽  
Valérie B Schini-Kerth
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Coronary Artery ◽  
Replicative Senescence ◽  
Critical Role ◽  
Primary Cultures ◽  
Enos Expression ◽  
Potent Inducer ◽  
In Cells

Endothelial cell senescence promotes endothelial dysfunction, which has been suggested to have a critical role in the initiation and/or progression of atherosclerosis, and also to contribute to the pathogenesis of age-associated vascular disorders. Endothelial senescence is characterized by an irreversible cell cycle arrest, which involves an increased activity of p53 and its downstream effector p21. Endothelial senescence is also associated with a decreased expression of endothelial nitric oxide synthase (eNOS). The present study has evaluated whether the Crataegus special extract WS ® 1442, a rich source of polyphenols and a potent inducer of eNOS activation, prevents replicative senescence of porcine coronary artery endothelial cells, and, if so, to elucidate the underlying mechanism. Replicative senescence was induced by sequential passaging of primary cultures of endothelial cells up to the fourth passage (P4). Changes of endothelial senescence were determined by measuring senescence-associated β-galactosidase (SA-β-gal) activity. Western blot was used to analyze the protein expression of p53, p21 and eNOS. Compared to P1, the SA-β-gal activity was 240% increased in cells at P4 ( P <0.001), and this effect was associated with 93% ( P <0.001) and 56% ( P <0.001) increased expression of p53, p21 and a 87% decreased expression of eNOS ( P <0.001). Treatment of P3 cells with the p53 inhibitor (pifithrin) reduced 43% SA-β-gal activity indicating a role of p53 activity in replicative senescence ( P <0.001). Treatment of endothelial cells with the Crataegus extract reduced by 56% the SA-β-gal activity ( P <0.01), improved by 131 % eNOS expression ( P <0.01) and reduced by 39% the up-regulation of p21 in cells at P4 without affecting the expression level of p53. The inhibitor of eNOS, L-NAME promoted the induction of endothelial senescence at P1 and reduced the inhibitory effect of the Crataegus extract on SA-β-gal activity at P3. In conclusion, the present findings indicate that the Crataegus extract delays endothelial cell replicative senescence most likely by preventing the downregulation of eNOS expression and activity and the upregulation of the p53/p21 pathway.

scholarly journals E3 Ubiquitin Ligase HOIP Attenuates Apoptotic Cell Death Induced by Cisplatin

2014 ◽  
Vol 74 (8) ◽  
pp. 2246-2257 ◽  
Author(s):  
Craig MacKay ◽  
Eilís Carroll ◽  
Adel F.M. Ibrahim ◽  
Amit Garg ◽  
Gareth J. Inman ◽  
...  
Keyword(s):  
Cell Death ◽  
Ubiquitin Ligase ◽  
Apoptotic Cell ◽  
E3 Ubiquitin Ligase ◽  
Apoptotic Cell Death
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