scholarly journals Regulation of mitophagy by the Gp78 E3 ubiquitin ligase

2013 ◽  
Vol 24 (8) ◽  
pp. 1153-1162 ◽  
Author(s):  
Min Fu ◽  
Pascal St-Pierre ◽  
Jay Shankar ◽  
Peter T. C. Wang ◽  
Bharat Joshi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
E3 Ubiquitin Ligase ◽  
Mitochondrial Fusion ◽  
Hek293 Cells ◽  
Chain Elongation ◽  
Mitochondrial Depolarization ◽  
Ubiquitin Ligase Activity ◽  
Sirna Knockdown

Glycoprotein 78 (Gp78) is a critical E3 ubiquitin ligase in endoplasmic reticulum–associated degradation. Overexpression of Flag-tagged Gp78 (Flag-gp78), but not Flag-gp78 mutated in its RING-finger domain (Flag-RINGmut) with deficient ubiquitin ligase activity, induces mitochondrial fragmentation and ubiquitination and proteasome-dependent degradation of the mitofusin (Mfn) mitochondrial fusion factors Mfn1/Mfn2. After mitochondrial depolarization with carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flag-gp78 induced a threefold loss of depolarized mitochondria and significant loss of the inner mitochondrial protein OxPhosV. Flag-gp78–dependent loss of OxPhosV, but not Mfn1 or Mfn2, was prevented by small interfering RNA (siRNA) knockdown of the autophagy protein Atg5 in CCCP-treated cells. Gp78-induced mitophagy required ubiquitin ligase activity, as it is not observed upon transfection of Flag-RINGmut or cotransfection of Flag-gp78 with ubiquitin mutated at three critical lysine residues (K29, 48, 63R) involved in polyubiquitin chain elongation. Short hairpin RNA knockdown of Gp78 in HT-1080 fibrosarcoma cells increased mitofusin levels and reduced depolarization-induced mitophagy, whereas siRNA knockdown showed that Mfn1, but not Mfn2, was required for Gp78-dependent depolarization-induced mitophagy. Mitochondrial depolarization induced Gp78-dependent expression of the autophagic marker LC3II and recruitment of enhanced green fluorescent protein–LC3 to the Gp78- and calnexin-labeled, mitochondria-associated ER. Finally, Gp78-induced mitophagy is Parkin independent, as it occurs in Parkin-null HeLa cells and upon siRNA-mediated Parkin knockdown in HEK293 cells. This study therefore describes a novel role for the ER-associated Gp78 ubiquitin ligase and the Mfn1 mitochondrial fusion factor in mitophagy.

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

2021 ◽  
Author(s):  
◽  
Carla Coppola
Keyword(s):  
Ubiquitin Ligase ◽  
Physcomitrella Patens ◽  
Fluorescent Protein ◽  
Storage Proteins ◽  
Higher Plants ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligases ◽  
Ubiquitin Ligase Activity ◽  
Vacuolar Transport

In this study, I focused on a new family of receptors, called RMRs (Receptor-like Membrane RING-H2) and I tried to investigate their role in the moss Physcomitrium patens Mitten (previously Physcomitrella patens). There is some evidence that in Angiosperms, RMRs are vacuolar receptors for the neutral/storage vacuole that is a compartment where storage proteins and metabolites are accumulated during seeds development or in somatic tissues. It is distinguished from lytic vacuole which has the same functions as animal lysosomes. The five PpRMR genes have been knocked-out, yielding viable material without visible phenotype (Ayachi, 2012). A trafficking phenotype was described by Fahr (2017) who generated the construct Citrine-Cardosin (Ci-Card) composed of the fluorescent protein Citrine fused to the C-terminal vacuolar sorting determinant (ctVSD) from cardosin A (cardosin is addressed to the vacuole in higher plants —Pereira et al., 2013). The fusion protein was delivered to the central vacuole of PpWT but mistargeted in PpRMR-KO lines, indicating that the targeting of this protein to the vacuole depends on PpRMRs. The introduction of this thesis presents the plant endomembrane system, with particular attention to vacuolar transport and ubiquitylation. In the second chapter, I show the techniques used to attempt to detect PpRMRs by Western Blot: our failure may be due to a rapid degradation of these proteins, which could prevent their detection. In the third chapter, I focused on PpRMR2 involvement in ubiquitylation. We hypothesize that PpRMRs are E3 ligases because they are members of the PA-TM-RING protein family. Most of these proteins have an E3 ubiquitin ligase activity in animals (Seroogy et al., 2004; Borchers et al., 2002), for this reason, we think that plant PpRMRs could have this function as well, which could contribute to vacuolar targeting. Indeed, I could confirm that PpRMR2 has an E3 ubiquitin ligase activity. PpRMRs substrates are still unknown in moss thus we have analysed putative candidates supposing that they could be ubiquitylated by PpRMRs. We have tested this hypothesis through in vitro ubiquitylation assays, obtaining ambiguous results. In the fourth chapter, I show preliminary results about the visible phenotype of PpRMR-KO mutants: PpWT and PpRMR-KO lines displayed phenotypic differences in leafy gametophores, which were accentuated upon salt stress exposure. Lastly, I transformed the transgenic lines PpWT/Ci-Card and Pp5KO/Ci-Card with mutated versions of PpRMR2 and analysed their effect on vacuolar transport by confocal microscopy. For most of the constructions tested, the trafficking was perturbed in both lines. Only PpWT/Ci-Card expressing PpRMR2ΔSer (lacking the Serine-Rich motif) displayed a typical vacuolar pattern.

scholarly journals The RBCC GeneRFP2(Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD

2007 ◽  
Vol 18 (5) ◽  
pp. 1670-1682 ◽  
Author(s):  
Mikael Lerner ◽  
Martin Corcoran ◽  
Diana Cepeda ◽  
Michael L. Nielsen ◽  
Roman Zubarev ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Yellow Fluorescent Protein ◽  
E3 Ubiquitin Ligase ◽  
Coiled Coil ◽  
Ring Finger ◽  
Ubiquitin Ligase Activity

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

Switching on ubiquitylation by phosphorylating a ubiquitous activator

2014 ◽  
Vol 460 (3) ◽  
pp. e1-e3 ◽  
Author(s):  
Gary S. Shaw
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Ubiquitin Ligase ◽  
Mitochondrial Protein ◽  
E3 Ubiquitin Ligase ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Depolarization ◽  
Biochemical Journal ◽  
Phosphatase And Tensin Homologue ◽  
Early Onset Parkinson’S Disease

The dysfunction of the E3 ubiquitin ligase Parkin is a key contributor to the development of early-onset Parkinson's disease. Parkin is responsible for the labelling of outer mitochondrial membrane proteins with the small modifier protein ubiquitin in response to oxidative stress. This ubiquitylation signals the clearance of the damaged mitochondria to preserve overall cell health. Recent structural and biochemical experiments have shown that native Parkin exists in an autoinhibited state that must be activated in order to unmask its full ubiquitylation potential. In a recent article in the Biochemical Journal (vol. 460, pp. 127–139), Kazlauskaite and co-workers identified that the Parkinson's disease-associated kinase PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1] can phosphorylate ubiquitin in response to mitochondrial depolarization. Furthermore, the authors demonstrated that phosphorylated ubiquitin can activate Parkin's E3 ligase activity and promote both increased autoubiquitylation and substrate ubiquitylation of the mitochondrial protein Miro1. The study provides exciting initial insights that show how PINK1 might activate ubiquitin through phosphorylation, and how this important regulatory step might switch on Parkin-mediated ubiquitylation.

scholarly journals Id1 enhances RING1b E3 ubiquitin ligase activity through the Mel-18/Bmi-1 polycomb group complex

Oncogene ◽  
2010 ◽  
Vol 29 (43) ◽  
pp. 5818-5827 ◽  
Author(s):  
T Qian ◽  
J-Y Lee ◽  
J-H Park ◽  
H-J Kim ◽  
G Kong
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Polycomb Group ◽  
Ubiquitin Ligase Activity

Post-translational regulation of FLC is mediated by an E3 ubiquitin ligase activity of SINAT5 in Arabidopsis

Plant Science ◽  
2007 ◽  
Vol 173 (2) ◽  
pp. 269-275 ◽  
Author(s):  
Bong Soo Park ◽  
Wan Gyu Sang ◽  
Song Yion Yeu ◽  
Yang Do Choi ◽  
Nam-Chon Paek ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Translational Regulation ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligase Activity

scholarly journals A novel role for BRCA1 in regulating breast cancer cell spreading and motility

2011 ◽  
Vol 192 (3) ◽  
pp. 497-512 ◽  
Author(s):  
Elisabeth D. Coene ◽  
Catarina Gadelha ◽  
Nicholas White ◽  
Ashraf Malhas ◽  
Benjamin Thomas ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Cell Spreading ◽  
Dominant Negative ◽  
Full Length ◽  
Suppressor Activity ◽  
Ubiquitin Ligase Activity ◽  
Mcf 7

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain–interacting proteins. Ezrin–radixin–moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA11634–1863 acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA11634–1863 in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.

scholarly journals E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins

2022 ◽  
Author(s):  
Grant Dewson ◽  
Alan Shuai Huang ◽  
Hui San Chin ◽  
Boris Reljic ◽  
Tirta M Djajawi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Apoptotic Cell ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligase Activity ◽  
A Genome ◽  
Library Screen ◽  
Important Regulator ◽  
Apoptotic Activity ◽  
Bh3 Mimetic ◽  
Survival Proteins

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

scholarly journals Inhibition of cIAP1 as a strategy for targeting c-MYC–driven oncogenic activity

2018 ◽  
Vol 115 (40) ◽  
pp. E9317-E9324 ◽  
Author(s):  
Haoyan Li ◽  
Yanjia Fang ◽  
Chunyi Niu ◽  
Hengyi Cao ◽  
Ting Mi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Pharmacological Agents ◽  
Protein Levels ◽  
Ubiquitin Ligase Activity ◽  
Molecule Inhibitor ◽  
Smac Mimetics ◽  
High Throughput Assay ◽  
In Cells

Protooncogenec-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.

scholarly journals Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism

2021 ◽  
Vol 320 (1) ◽  
pp. C45-C56
Author(s):  
David C. Hughes ◽  
Daniel C. Turner ◽  
Leslie M. Baehr ◽  
Robert A. Seaborne ◽  
Mark Viggars ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Tibialis Anterior Muscle ◽  
E3 Ubiquitin Ligase ◽  
Skeletal Muscle Atrophy ◽  
The Impact

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5’s role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3β/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (−9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3β activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (−4.6%) and larger reductions in fiber CSA (−18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

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