Parkin is an E3 ubiquitin-ligase for normal and mutant ataxin-2 and prevents ataxin-2-induced cell death

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.

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Abstract 243: The E3 Ubiquitin Ligase Parkin Mediates Clearance of Damaged Mitochondria via Two Distinct Pathways

Circulation Research ◽

10.1161/res.117.suppl_1.243 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Babette C Hammerling ◽

Melissa Q Cortez ◽

Rita A Hanna ◽

Eileen R Gonzalez ◽

Åsa B Gustafsson

Keyword(s):

Cell Death ◽

Ubiquitin Ligase ◽

Ischemia Reperfusion ◽

E3 Ubiquitin Ligase ◽

Dominant Negative ◽

Mitochondrial Ros ◽

Late Endosomes ◽

Early Endosomes ◽

Important Regulator ◽

Rapid Activation

Damaged mitochondria release reactive oxygen species and pro-death factors which can lead to loss of cardiac myocytes. To protect against such damage, myocytes have developed several mechanisms of quality control that act both on the protein and organelle levels. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin can also mediate clearance of mitochondria via the endosomal-lysosomal pathway. We found that Parkin promotes clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Mitochondrial damage leads to rapid activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. We further observed increased activation of Rab5, a protein involved in early endosome formation, in both WT and Atg5-/- MEFs after treatment with FCCP. In addition, we observed sequestration of damaged mitochondria in Rab5+ and Rab7+ early and late endosomes, respectively. Mitochondria also colocalized with Lamp2+ vesicles in Atg5-/- MEFs indicating that the mitochondria are ultimately being delivered to the lysosomes for degradation. Overexpression of Rab5S34N, a dominant negative of Rab5, reduces FCCP-mediated clearance and increases cell death in Atg5-/- MEFs. Pharmacological inhibition of the endosomal-lysosomal pathway also results in increased FCCP-mediated cell death. Furthermore, we confirmed that FCCP treatment or simulated ischemia reperfusion exposure induces Rab5 activation with subsequent mitochondrial sequestration in early endosomes in neonatal myocytes. Interestingly, the activation of Rab5 is abrogated in the presence of the mitochondrial targeted antioxidant Mito-Tempo, suggesting that mitochondrial ROS is involved in the activation the endosomal pathway. Mitochondrial clearance via this pathway is also dependent on Parkin, as FCCP treatment fails to activate Rab5 and induce mitochondrial clearance in both WT and Atg5-/- MEFS in the absence of Parkin. Thus, our data suggest that Parkin can mediate clearance of damaged mitochondria via two distinct pathways in cells.

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Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activity

The Plant Cell ◽

10.1105/tpc.104.025171 ◽

2004 ◽

Vol 16 (10) ◽

pp. 2795-2808 ◽

Cited By ~ 225

Author(s):

Li-Rong Zeng ◽

Shaohong Qu ◽

Alicia Bordeos ◽

Chengwei Yang ◽

Marietta Baraoidan ◽

...

Keyword(s):

Cell Death ◽

Plant Cell ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Negative Regulator ◽

Repeat Protein ◽

Ubiquitin Ligase Activity ◽

Armadillo Repeat

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The Pepper E3 Ubiquitin Ligase RING1 Gene, CaRING1, Is Required for Cell Death and the Salicylic Acid-Dependent Defense Response

PLANT PHYSIOLOGY ◽

10.1104/pp.111.177568 ◽

2011 ◽

Vol 156 (4) ◽

pp. 2011-2025 ◽

Cited By ~ 65

Author(s):

Dong Hyuk Lee ◽

Hyong Woo Choi ◽

Byung Kook Hwang

Keyword(s):

Cell Death ◽

Salicylic Acid ◽

Ubiquitin Ligase ◽

Defense Response ◽

E3 Ubiquitin Ligase

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606742113 ◽

2016 ◽

Vol 113 (37) ◽

pp. 10394-10399 ◽

Cited By ~ 16

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.

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

10.1101/2021.07.21.453219 ◽

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.

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