Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Download Full-text

Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

Circulation Research ◽

10.1161/res.109.suppl_1.ap180 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Kedryn K Baskin ◽

Rebecca Salazar ◽

Wenhao Chen ◽

Heinrich Taegtmeyer

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Fold Increase ◽

Ubiquitin Proteasome System ◽

Initiation Factor ◽

Skeletal Muscle Atrophy ◽

Heart Weight ◽

Cross Sectional

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Download Full-text

mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1521919112 ◽

2015 ◽

Vol 112 (52) ◽

pp. 15790-15797 ◽

Cited By ~ 185

Author(s):

Jinghui Zhao ◽

Bo Zhai ◽

Steven P. Gygi ◽

Alfred Lewis Goldberg

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Protein Degradation ◽

Mammalian Cells ◽

Cholesterol Biosynthesis ◽

Mammalian Target Of Rapamycin ◽

Ubiquitin Proteasome System ◽

Mtor Inhibition ◽

Protein Ubiquitination ◽

Ubiquitin Proteasome

Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440–443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth.

Download Full-text

Early activation of ubiquitin-proteasome system at the diaphragm tissue occurs independently of left ventricular dysfunction in SHR rats

Experimental Biology and Medicine ◽

10.1177/1535370219897883 ◽

2020 ◽

Vol 245 (3) ◽

pp. 245-253

Author(s):

Pamella Ramona Moraes de Souza ◽

Renata Kelly da Palma ◽

Rodolfo Paula Vieira ◽

Fernando dos Santos ◽

Wilson Max Almeida Monteiro-De-Moraes ◽

...

Keyword(s):

Protein Degradation ◽

Autonomic Dysfunction ◽

Ubiquitin Proteasome System ◽

Left Ventricular ◽

Diaphragm Muscle ◽

Respiratory Pattern ◽

Type I ◽

Cross Sectional ◽

Ventilatory Pattern ◽

Ubiquitin Proteasome

Hypertensive status induces modifications in the respiratory profile. Previous studies have indicated that hypertensive rats show increased respiratory-sympathetic coupling compared to normotensive rats. However, these effects and especially the mechanisms underlying such effects are not well known. Thus, we evaluated the influence of high blood pressure and autonomic dysfunction on a ventilatory pattern associated with lung injury and on the ubiquitin-proteasome system of the diaphragm muscle. Autonomic cardiovascular modulation (systolic BP variance and low-frequency band and pulse interval variance) and arterial blood gases patterns (pH, pO2, HCO3, SpO2), can be changed by hypertension, as well exacerbated chemoreflex pressor response. We observed that the diaphragm muscle of SHR showed increase in type I cross-sectional fiber (16%) and reduction in type II cross-sectional fiber area (41%), increased activity of the ubiquitin-proteasome system and lipid peroxidation, with no differences between groups in the analysis of ubiquitinated proteins and misfolded proteins. Our results showed that hypertension induced functional compensatory/adverse alterations associated with diaphragm fiber type changes and protein degradation as well as changed autonomic control of circulation. In conclusion, we believe there is an adaptation in ventilatory pattern in regarding to prevent the development of fatigue and muscle weakness and improve ventilatory endurance. Impact statement It was well known that hypertension can be driven by increased sympathetic activity and has been documented as a central link between autonomic dysfunction and alterations in the respiratory pattern. Our study demonstrated the impact of hypertension in ventilatory mechanics and their relationship with diaphragm muscle protein degradation. These findings may assist us in future alternative treatments to prevent diaphragm fatigue and weakness in hypertensive patients.

Download Full-text

Ubiquitin Ligases at the Heart of Skeletal Muscle Atrophy Control

Molecules ◽

10.3390/molecules26020407 ◽

2021 ◽

Vol 26 (2) ◽

pp. 407

Author(s):

Dulce Peris-Moreno ◽

Laura Cussonneau ◽

Lydie Combaret ◽

Cécile Polge ◽

Daniel Taillandier

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Signaling Pathways ◽

Muscle Atrophy ◽

Ubiquitin Ligases ◽

Ubiquitin Proteasome System ◽

Skeletal Muscle Atrophy ◽

Mortality And Morbidity ◽

E3 Ligases ◽

Ubiquitin Proteasome

Skeletal muscle loss is a detrimental side-effect of numerous chronic diseases that dramatically increases mortality and morbidity. The alteration of protein homeostasis is generally due to increased protein breakdown while, protein synthesis may also be down-regulated. The ubiquitin proteasome system (UPS) is a master regulator of skeletal muscle that impacts muscle contractile properties and metabolism through multiple levers like signaling pathways, contractile apparatus degradation, etc. Among the different actors of the UPS, the E3 ubiquitin ligases specifically target key proteins for either degradation or activity modulation, thus controlling both pro-anabolic or pro-catabolic factors. The atrogenes MuRF1/TRIM63 and MAFbx/Atrogin-1 encode for key E3 ligases that target contractile proteins and key actors of protein synthesis respectively. However, several other E3 ligases are involved upstream in the atrophy program, from signal transduction control to modulation of energy balance. Controlling E3 ligases activity is thus a tempting approach for preserving muscle mass. While indirect modulation of E3 ligases may prove beneficial in some situations of muscle atrophy, some drugs directly inhibiting their activity have started to appear. This review summarizes the main signaling pathways involved in muscle atrophy and the E3 ligases implicated, but also the molecules potentially usable for future therapies.

Download Full-text

From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation

Frontiers in Chemistry ◽

10.3389/fchem.2021.672267 ◽

2021 ◽

Vol 9 ◽

Author(s):

Carlotta Cecchini ◽

Sara Pannilunghi ◽

Sébastien Tardy ◽

Leonardo Scapozza

Keyword(s):

Molecular Weight ◽

Drug Discovery ◽

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Cell Permeability ◽

Polar Surface Area ◽

Ubiquitin Proteasome ◽

Cellular Permeability ◽

Related Proteins

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional degraders that specifically eliminate targeted proteins by hijacking the ubiquitin-proteasome system (UPS). This modality has emerged as an orthogonal approach to the use of small-molecule inhibitors for knocking down classic targets and disease-related proteins classified, until now, as “undruggable.” In early 2019, the first targeted protein degraders reached the clinic, drawing attention to PROTACs as one of the most appealing technology in the drug discovery landscape. Despite these promising results, PROTACs are often affected by poor cellular permeability due to their high molecular weight (MW) and large exposed polar surface area (PSA). Herein, we report a comprehensive record of PROTAC design, pharmacology and thermodynamic challenges and solutions, as well as some of the available strategies to enhance cellular uptake, including suggestions of promising biological tools for the in vitro evaluation of PROTACs permeability toward successful protein degradation.

Download Full-text

Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽

10.3390/biom11010110 ◽

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.

Download Full-text

DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.254 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii63-ii63

Author(s):

Lakshmi Bollu ◽

Derek Wainwright ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

...

Keyword(s):

Protein Degradation ◽

Time Course ◽

Immune Cell ◽

Enzyme Inhibitors ◽

Tumor Development ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Specific Protein ◽

Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

Download Full-text

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

Frontiers in Plant Science ◽

10.3389/fpls.2021.806129 ◽

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.

Download Full-text

Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

10.1101/2020.03.30.015370 ◽

2020 ◽

Author(s):

Ganapathi Kandasamy ◽

Ashis Kumar Pradhan ◽

R Palanimurugan

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Rna Degradation ◽

26S Proteasome ◽

Cellular Proteins ◽

Cellular Homeostasis ◽

Substrate Degradation ◽

Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

Download Full-text