scholarly journals Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay

2018 ◽  
Author(s):  
Joanna Koszela ◽  
Nhan T Pham ◽  
David Evans ◽  
Stefan Mann ◽  
Irene Perez-Pi ◽  
...  
Keyword(s):  
Real Time ◽  
Ubiquitin Proteasome System ◽  
Enzymatic Activities ◽  
Confocal Imaging ◽  
Experimental Compartment ◽  
Ubiquitin Proteasome ◽  
Real Time Tracking ◽  
The Stability ◽  
Enzymatic Pathways

AbstractThe ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labelled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2 and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. As a sensitive, quantitative, flexible and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

scholarly journals A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

2022 ◽  
Vol 12 ◽  
Author(s):  
Aida Kozlic ◽  
Nikola Winter ◽  
Theresia Telser ◽  
Jakob Reimann ◽  
Katrin Rose ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Functional Assay ◽  
The Novel ◽  
In Planta ◽  
Amino Terminal ◽  
Weak Binding ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Proteasome ◽  
The Stability

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

scholarly journals The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1

2011 ◽  
Vol 194 (1) ◽  
pp. 17-25 ◽  
Author(s):  
Rachel Lander ◽  
Kara Nordin ◽  
Carole LaBonne
Keyword(s):  
Structural Diversity ◽  
Ubiquitin Proteasome System ◽  
Common Mechanism ◽  
Rich Domain ◽  
Transcriptional Regulatory ◽  
Snail Family ◽  
Ubiquitin Proteasome ◽  
Snail Proteins ◽  
The Stability ◽  
F Box Protein

A small group of core transcription factors, including Twist, Snail, Slug, and Sip1, control epithelial–mesenchymal transitions (EMTs) during both embryonic development and tumor metastasis. However, little is known about how these factors are coordinately regulated to mediate the requisite behavioral and fate changes. It was recently shown that a key mechanism for regulating Snail proteins is by modulating their stability. In this paper, we report that the stability of Twist is also regulated by the ubiquitin–proteasome system. We found that the same E3 ubiquitin ligase known to regulate Snail family proteins, Partner of paired (Ppa), also controlled Twist stability and did so in a manner dependent on the Twist WR-rich domain. Surprisingly, Ppa could also target the third core EMT regulatory factor Sip1 for proteasomal degradation. Together, these results indicate that despite the structural diversity of the core transcriptional regulatory factors implicated in EMT, a common mechanism has evolved for controlling their stability and therefore their function.

scholarly journals Killing by Degradation: Regulation of Apoptosis by the Ubiquitin-Proteasome-System

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3465
Author(s):  
Ruqaia Abbas ◽  
Sarit Larisch
Keyword(s):  
Apoptotic Cell ◽  
Ubiquitin Proteasome System ◽  
Cancer Therapies ◽  
E3 Ligases ◽  
Ubiquitin Proteasome ◽  
A Cell ◽  
Positive And Negative Feedback ◽  
Apoptotic Proteins ◽  
The Stability ◽  
Cell Suicide

Apoptosis is a cell suicide process that is essential for development, tissue homeostasis and human health. Impaired apoptosis is associated with a variety of human diseases, including neurodegenerative disorders, autoimmunity and cancer. As the levels of pro- and anti-apoptotic proteins can determine the life or death of cells, tight regulation of these proteins is critical. The ubiquitin proteasome system (UPS) is essential for maintaining protein turnover, which can either trigger or inhibit apoptosis. In this review, we will describe the E3 ligases that regulate the levels of pro- and anti-apoptotic proteins and assisting proteins that regulate the levels of these E3 ligases. We will provide examples of apoptotic cell death modulations using the UPS, determined by positive and negative feedback loop reactions. Specifically, we will review how the stability of p53, Bcl-2 family members and IAPs (Inhibitor of Apoptosis proteins) are regulated upon initiation of apoptosis. As increased levels of oncogenes and decreased levels of tumor suppressor proteins can promote tumorigenesis, targeting these pathways offers opportunities to develop novel anti-cancer therapies, which act by recruiting the UPS for the effective and selective killing of cancer cells.

scholarly journals Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages

Biology Open ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. bio051029 ◽  
Author(s):  
Maya Morita ◽  
Mayu Kajiye ◽  
Chiye Sakurai ◽  
Shuichi Kubo ◽  
Miki Takahashi ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Ubiquitin Proteasome System ◽  
Snare Proteins ◽  
Regulatory Function ◽  
Limiting Factor ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Ubiquitin Proteasome ◽  
Syntaxin 11

ABSTRACTMicrotubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.

Clinical Relevance of Ubiquitin-Proteasome System Profiling in Acute Leukemias.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2633-2633
Author(s):  
Wanlong Ma ◽  
Hagop M. Kantarjian ◽  
XI Zhang ◽  
Xiuqiang Wang ◽  
Zeev Estrov ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ubiquitin Proteasome System ◽  
Enzymatic Activities ◽  
Acute Leukemias ◽  
Clinical Behavior ◽  
Protein Levels ◽  
Control Subjects ◽  
Ubiquitin Proteasome ◽  
Proteasome Protein

Abstract Abstract 2633 Poster Board II-609 The ubiquitin-proteasome system (UPS) plays a major role in the homeostasis of cellular proteins: chymotrypsin-like (Ch-L), caspase-like (Cas-L), and trypsin-like (Tr-L). The UPS system is involved in most critical cellular processes and its role in oncogenesis is well established. Here we compared UPS protein levels (proteasome and ubiquitin) and proteasome enzymatic activities in peripheral blood plasma from newly diagnosed patients with acute myeloid leukemia (AML; n=147), acute lymphoblastic leukemia (ALL; n=34?), or myelodysplastic syndrome (MDS ; n=27), and 99 apparently healthy control subjects. Proteasome and ubiquitin were measured using immunoassays based on electro-chemiluminescence technology. The proteasome enzymatic activities were measure using a standard enzymatic assay utilizing fluorogenic peptide-AMC substrate. By normalizing these enzymatic activities to the levels of proteasome protein in plasma, we also determined specific proteasome enzyme activities (Ch-L/p, Cas-L/p, and Tr-L, respectively). AML, ALL, and MDS patients all had high levels of proteasome protein, ubiquitin, and enzymatic activities relative to control subjects. However, each specific enzyme activity in general was lower in all 3 disease groups than in control subjects, which suggests that each proteasome has lower enzymatic activity in blast cells but we cannot rule out that the number of proteasomes might be increased. Despite these similarities, AML, ALL, and MDS each exhibited a specific profile of UPS protein and enzymatic activities . Proteasome protein levels and enzymatic activities also correlated with clinical behavior. In AML, proteasome protein level was a strong predictor of survival as a both a continuous (P<0.00001) and a dichotomous (P=0.04) variable, independent of cytogenetics, performance status, and age. In ALL, Ch-L/p showed a significant negative correlation with survival (P=0.0015). In conclusion, these data demonstrate that each disease has a unique UPS profile and confirm that the UPS system plays a major role in the leukemic process. In addition, profiling the UPS in leukemias using plasma provides valuable biomarkers that can be used to help predict clinical behavior and may ultimately help manage disease. Disclosures: No relevant conflicts of interest to declare.

The ubiquitin–proteasome system regulates the stability and activity of the glucose sensor glucokinase in pancreatic β-cells

2013 ◽  
Vol 456 (2) ◽  
pp. 173-184 ◽  
Author(s):  
Anke Hofmeister-Brix ◽  
Sigurd Lenzen ◽  
Simone Baltrusch
Keyword(s):  
Protein Misfolding ◽  
Glucose Sensor ◽  
Ubiquitin Proteasome System ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Insulin Secreting Cells ◽  
Ubiquitin Proteasome ◽  
The Stability

The glucose phosphorylating enzyme glucokinase is regulated by the ubiquitin–proteasome system. Inhibition of the proteasome leads to reduced glucokinase activity, glucokinase protein misfolding and localization of glucokinase in aggresomes in insulin-secreting cells, pancreatic β-cells and hepatocytes.

Proteomic Characterization of Aggregating Proteins after the Inhibition of the Ubiquitin Proteasome System

2011 ◽  
Vol 10 (3) ◽  
pp. 1062-1072 ◽  
Author(s):  
Inga B. Wilde ◽  
Maria Brack ◽  
Jason M. Winget ◽  
Thibault Mayor
Keyword(s):  
Ubiquitin Proteasome System ◽  
Ubiquitin Proteasome

scholarly journals Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

2012 ◽  
Vol 448 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jonas Boehringer ◽  
Christiane Riedinger ◽  
Konstantinos Paraskevopoulos ◽  
Eachan O. D. Johnson ◽  
Edward D. Lowe ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Functional Characterization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Structural Motif ◽  
Protein Protein Interactions ◽  
Ubiquitin Proteasome

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

The Emerging Role of CSN6 in Biological Behavior and Cancer Progress

2019 ◽  
Vol 19 (10) ◽  
pp. 1198-1204
Author(s):  
Zun Mao ◽  
Cheng Chen ◽  
Dong-Sheng Pei
Keyword(s):  
Clinical Care ◽  
Protein Molecule ◽  
Ubiquitin Proteasome System ◽  
Biological Behavior ◽  
Developmental Defects ◽  
Normal Tissues ◽  
Wide Range ◽  
Ubiquitin Proteasome ◽  
Regulatory Capacity ◽  
The Stability

Background:The Constitutive Photomorphogenesis 9 (COP9) signalosome (CSN) subunit 6 (CSN6) noticeably acts as a regulator of the degradation of cancer-related proteins, which contributes to cancerogenesis. The aims of this paper are to expound the research advances of CSN6, particularly focusing on roles of CSN6 in the regulation of biological behavior and cancer progress.Methods:Literature from PubMed and Web of Science databases about biological characteristics and application of CSN6 published in recent years was collected to conduct a review.Results:CSN6, not only the non-catalytic Mpr1p and Pad1p N-terminal (MPN) subunit of CSN, but also a relatively independent protein molecule, has received great attention as a regulator of a wide range of developmental processes by taking part in the ubiquitin-proteasome system and signal transduction, as well as regulating genome integrity and DNA damage response. In addition, phosphorylation of CSN6 increases the stability of CSN6, thereby promoting its regulatory capacity. Moreover, CSN6 is overexpressed in many types of cancer compared with normal tissues and is involved in the regulation of several important intracellular pathways, consisting of cell proliferation, migration, invasion, transformation, and tumorigenesis.Conclusion:We mainly present insights into the function and research development of CSN6, hoping that it can help guide the treatment of developmental defects and improve clinical care, especially in the regulation of cancer signaling pathways.

scholarly journals Mutant Ubiquitin-Mediated β-Secretase Stability via Activation of Caspase-3 is Related to β-Amyloid Accumulation in Ischemic Striatum in Rats

2009 ◽  
Vol 30 (3) ◽  
pp. 566-575 ◽  
Author(s):  
Yong Zhang ◽  
Man Xiong ◽  
Ri-Qiang Yan ◽  
Feng-Yan Sun
Keyword(s):  
Caspase 3 ◽  
Ubiquitin Proteasome System ◽  
Artery Occlusion ◽  
Amyloid Accumulation ◽  
Ipsilateral Striatum ◽  
Ubiquitin Proteasome ◽  
Amyloid Aβ ◽  
Β Amyloid ◽  
The Stability ◽  
Cerebral Artery Occlusion

Previous studies have demonstrated that ischemic stroke increases β-amyloid (Aβ) production by increasing β-secretase (BACE1) through activation of caspase-3, and stimulates generation of mutant ubiquitin (UBB+1) in rat brains. In this study, we examined whether caspase-3 activation participates in the regulation of UBB+1 generation and UBB+1-mediated BACE1 stability in ischemic injured brains. The results showed that UBB+1 and activated caspase-3-immunopositive-stained cells were time dependently increased in the ipsilateral striatum of rat brains after middle cerebral artery occlusion. UBB+1-immunopositive cells could be co-stained with caspase-3, Aβ (UBB+1–Aβ), and BACE1 (UBB+1–BACE1). BACE1 protein could also be pulled down by immunoprecipitation with UBB+1 antibody. Z-DEVD-FMK (DEVD), a caspase-3 inhibitor, significantly decreased the level of UBB+1 protein and the number of UBB+1–Aβ and UBB+1–BACE1 double-stained cells in the ischemic striatum, as well as the level of UBB+1/BACE1 protein complex. We conclude that activation of caspase-3 might be upstream of UBB+1 formation and that excessive UBB+1 could bind to BACE1 and increase the stability of BACE1, thereby increasing Aβ in ischemic injured brains. These results suggest new biological and pathological effects of caspases and regulation of the ubiquitin–proteasome system in the brain. Our results provide new therapeutic targets to prevent further neurodegeneration in patients after stroke.

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