scholarly journals A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination

2021 ◽  
Vol 26 (4) ◽  
pp. 547-559
Author(s):  
Ellen F. Vieux ◽  
Roman V. Agafonov ◽  
Lydia Emerson ◽  
Marta Isasa ◽  
Richard W. Deibler ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Ubiquitin Proteasome System ◽  
Binary Complex ◽  
Discovery Process ◽  
Drug Discovery Process ◽  
In Vitro System ◽  
Ubiquitin Proteasome ◽  
Catalytic Rate ◽  
Kinetics Of

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin–proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

scholarly journals Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 110
Author(s):  
Dina Aweida ◽  
Shenhav Cohen
Keyword(s):  
Protein Quality ◽  
Protein Structures ◽  
Ubiquitin Proteasome System ◽  
Surface Proteins ◽  
Catalytic Core ◽  
Complex Protein ◽  
Ubiquitin Proteasome ◽  
Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

scholarly journals Increasing autophagy does not affect neurogenic muscle atrophy

2018 ◽  
Vol 28 (3) ◽  
Author(s):  
Eva Pigna ◽  
Krizia Sanna ◽  
Dario Coletti ◽  
Zhenlin Li ◽  
Ara Parlakian ◽  
...  
Keyword(s):  
Muscle Mass ◽  
Muscle Atrophy ◽  
Ubiquitin Proteasome System ◽  
Autophagic Flux ◽  
Molecular Pathways ◽  
Muscle Loss ◽  
Relative Contribution ◽  
Ubiquitin Proteasome ◽  
Muscle Mass Loss ◽  
Kinetics Of

Physiological autophagy plays a crucial role in the regulation of muscle mass and metabolism, while the excessive induction or the inhibition of the autophagic flux contributes to the progression of several diseases. Autophagy can be activated by different stimuli, including cancer, exercise, caloric restriction and denervation. The latter leads to muscle atrophy through the activation of catabolic pathways, i.e. the ubiquitin-proteasome system and autophagy. However, the kinetics of autophagy activation and the upstream molecular pathways in denervated skeletal muscle have not been reported yet. In this study, we characterized the kinetics of autophagic induction, quickly triggered by denervation, and report the Akt/mTOR axis activation. Besides, with the aim to assess the relative contribution of autophagy in neurogenic muscle atrophy, we triggered autophagy with different stimuli along with denervation, and observed that four week-long autophagic induction, by either intermitted fasting or rapamycin treatment, did not significantly affect muscle mass loss. We conclude that: i) autophagy does not play a major role in inducing muscle loss following denervation; ii) nonetheless, autophagy may have a regulatory role in denervation induced muscle atrophy, since it is significantly upregulated as early as eight hours after denervation; iii) Akt/mTOR axis, AMPK and FoxO3a are activated consistently with the progression of muscle atrophy, further highlighting the complexity of the signaling response to the atrophying stimulus deriving from denervation.

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 Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

2020 ◽  
Vol 21 (11) ◽  
pp. 4151
Author(s):  
Lucie Tumova ◽  
Michal Zigo ◽  
Peter Sutovsky ◽  
Marketa Sedmikova ◽  
Pavla Postlerova
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Protein Composition ◽  
Surface Protein ◽  
Ubiquitin Proteasome System ◽  
Sperm Capacitation ◽  
Sperm Surface ◽  
Ubiquitin Proteasome ◽  
Seminal Plasma Proteins

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

scholarly journals USP14 Inhibition Regulates Tumorigenesis by Inducing Autophagy in Lung Cancer In Vitro

2019 ◽  
Vol 20 (21) ◽  
pp. 5300 ◽  
Author(s):  
Kyung Ho Han ◽  
Minseok Kwak ◽  
Tae Hyeong Lee ◽  
Min-soo Park ◽  
In-ho Jeong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Ubiquitin Proteasome System ◽  
Migration And Invasion ◽  
Deubiquitinating Enzyme ◽  
Lung Tumorigenesis ◽  
Ubiquitin Chain ◽  
Specific Protease ◽  
Ubiquitin Proteasome ◽  
Autophagy Pathway

The ubiquitin–proteasome system is an essential regulator of several cellular pathways involving oncogenes. Deubiquitination negatively regulates target proteins or substrates linked to both hereditary and sporadic forms of cancer. The deubiquitinating enzyme ubiquitin-specific protease 14 (USP14) is associated with proteasomes where it trims the ubiquitin chain on the substrate. Here, we found that USP14 is highly expressed in patients with lung cancer. We also demonstrated that USP14 inhibitors (IU1-47 and siRNA-USP14) significantly decreased cell proliferation, migration, and invasion in lung cancer. Remarkably, we found that USP14 negatively regulates lung tumorigenesis not only through apoptosis but also through the autophagy pathway. Our findings suggest that USP14 plays a crucial role in lung tumorigenesis and that USP14 inhibitors are potent drugs in lung cancer treatment.

Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

2012 ◽  
Vol 443 (3) ◽  
pp. 681-689 ◽  
Author(s):  
Wan Ning Vanessa Chow ◽  
Hon Wing Luk ◽  
Ho Yin Edwin Chan ◽  
Kwok-Fai Lau
Keyword(s):  
Cell Death ◽  
Degradation Mechanism ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Adaptor Protein ◽  
Ubiquitin Proteasome System ◽  
Exon 1 ◽  
Ubiquitin Proteasome

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW–polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.

scholarly journals NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

Archaea ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-14
Author(s):  
Ekaterina Yu. Bezsudnova ◽  
Tatiana E. Petrova ◽  
Natalia V. Artemova ◽  
Konstantin M. Boyko ◽  
Ivan G. Shabalin ◽  
...  
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Sites ◽  
Ternary Complex ◽  
Binary Complex ◽  
Relay Systems ◽  
Binary Complexes ◽  
Proton Relay ◽  
Functional Features

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

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