scholarly journals Neuronal Proteostasis is mediated by the switch-like expression of Heme-regulated Kinase 1, acting as both a sensor and effector

2019 ◽  
Author(s):  
Beatriz Alvarez-Castelao ◽  
Susanne tom Dieck ◽  
Claudia M. Fusco ◽  
Paul G. Donlin-Asp ◽  
Julio D. Perez ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Kinase Inhibitor ◽  
Neuronal Cell ◽  
Proteasome Inhibition ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Major Protein ◽  
Early Protein ◽  
Constitutively Active

AbstractAll cells, including neurons, have regulatory feedback mechanisms that couple protein synthesis and degradation to maintain and optimize protein concentrations in the face of intra- and extracellular perturbations. We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in neurons. When protein degradation by the UPS was inhibited we observed a coordinate dramatic reduction in nascent protein synthesis in both neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2a, surprisingly mediated by eIF2a kinase 1, or heme-regulated kinase inhibitor (HRI), known for its sensitivity to heme levels in erythrocyte precursors (Han et al., 2001). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of the short-lived HRI protein and enhanced translation via the increased availability of tRNAs for rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2a phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for HRI in neurons, acting as an “immediate early protein” that senses and responds to compromised function of the proteasome to restore proteostasis.One sentence summaryProteasome inhibition leads to a compensatory reduction in neuronal protein synthesis via the stabilization and enhanced translation of short-lived HRI kinase, which is constitutively active upon expression owing to low neuronal heme levels.

scholarly journals The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Beatriz Alvarez-Castelao ◽  
Susanne tom Dieck ◽  
Claudia M Fusco ◽  
Paul Donlin-Asp ◽  
Julio D Perez ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Kinase Inhibitor ◽  
Neuronal Cell ◽  
Proteasome Inhibition ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Major Protein ◽  
Protein Levels ◽  
Eif2α Phosphorylation

We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in rat and mouse neurons. When protein degradation was inhibited, we observed a coordinate dramatic reduction in nascent protein synthesis in neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2α, surprisingly mediated by eIF2α kinase 1, or heme-regulated kinase inhibitor (HRI). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of HRI and enhanced translation, likely via the increased availability of tRNAs for its rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2α phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for neuronal HRI that senses and responds to compromised function of the proteasome to restore proteostasis.

Ubiquitin-Dependent Proteolysis in Neurons

Physiology ◽  
2003 ◽  
Vol 18 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Lars Klimaschewski
Keyword(s):  
Neurodegenerative Diseases ◽  
Protein Degradation ◽  
Current Knowledge ◽  
Neurological Diseases ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Present Review ◽  
Major Protein ◽  
Ubiquitin Proteasome ◽  
The Brain

Various studies identified the ubiquitin-proteasome system as the prime suspect in causing neurodegenerative diseases. The present review summarizes our current knowledge about the expression, regulation, and functions of this major protein degradation pathway in the brain, with particular reference to the pathogenesis of associated neurological diseases.

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

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.

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

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.

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

2015 ◽  
Vol 112 (52) ◽  
pp. 15790-15797 ◽  
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.

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

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kedryn K Baskin ◽  
Rebecca Salazar ◽  
Wenhao Chen ◽  
Heinrich Taegtmeyer
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Skeletal Muscle Atrophy ◽  
Heart Weight ◽  
Cross Sectional ◽  
Mechanical Unloading ◽  
Ubiquitin Proteasome ◽  
Specific Proteins

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.

Targeting Protein Degradation in Cancer Treatment

2020 ◽  
Vol 14 ◽  
Author(s):  
Imane Bjij ◽  
Ismail Hdoufane ◽  
Mahmoud Soliman ◽  
Menče Najdoska-Bogdanov ◽  
Driss Cherqaoui
Keyword(s):  
Protein Degradation ◽  
Therapeutic Potential ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Cellular Protein ◽  
Cellular Level ◽  
Cancerous Cell ◽  
Promising Target ◽  
Anti Cancer ◽  
Ubiquitin Proteasome

: The ubiquitin proteasome system (UPS) is a crucial protein degradation pathway that involves several enzymes to maintain cellular protein homeostasis. This system has emerged as a major drug target against certain types of cancer as a disruption at the cellular level of UPS enzyme components forces the transformation of normal cell into cancerous cell. Although enormous advancements have been achieved in the understanding of tumorigenesis, efficient cancer therapy remains a goal towards alleviating this serious health issue. Since UPS has become a promising target for anticancer therapies, herein we provide comprehensive review of the ubiquitin proteasome system as a significant process for protein degradation. Herein, the anti-cancer therapeutic potential of this pathway is also discussed.

The Ubiquitin-Proteasome System and Memory: Moving Beyond Protein Degradation

2018 ◽  
Vol 24 (6) ◽  
pp. 639-651 ◽  
Author(s):  
Timothy J. Jarome ◽  
Rishi K. Devulapalli
Keyword(s):  
Protein Degradation ◽  
Chromatin Remodeling ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Memory Formation ◽  
Consolidation Process ◽  
Future Studies ◽  
Cellular Models ◽  
Ubiquitin Proteasome

Cellular models of memory formation have focused on the need for protein synthesis. Recently, evidence has emerged that protein degradation mediated by the ubiquitin-proteasome system (UPS) is also important for this process. This has led to revised cellular models of memory formation that focus on a balance between protein degradation and synthesis. However, protein degradation is only one function of the UPS. Studies using single-celled organisms have shown that non-proteolytic ubiquitin-proteasome signaling is involved in histone modifications and DNA methylation, suggesting that ubiquitin and the proteasome can regulate chromatin remodeling independent of protein degradation. Despite this evidence, the idea that the UPS is more than a protein degradation pathway has not been examined in the context of memory formation. In this article, we summarize recent findings implicating protein degradation in memory formation and discuss various ways in which both ubiquitin signaling and the proteasome could act independently to regulate epigenetic-mediated transcriptional processes necessary for learning-dependent synaptic plasticity. We conclude by proposing comprehensive models of how non-proteolytic functions of the UPS could work in concert to control epigenetic regulation of the cellular memory consolidation process, which will serve as a framework for future studies examining the role of the UPS in memory formation.

scholarly journals Autophagy enhances memory erasure through synaptic destabilization

2017 ◽  
Author(s):  
Mohammad Shehata ◽  
Kareem Abdou ◽  
Kiriko Choko ◽  
Mina Matsuo ◽  
Hirofumi Nishizono ◽  
...  
Keyword(s):  
Anxiety Disorders ◽  
Protein Degradation ◽  
Degradation Pathway ◽  
Male Rats ◽  
Major Protein ◽  
Synaptic Protein ◽  
Male Mice ◽  
Therapeutic Tool ◽  
Contextual Memory ◽  
Autophagy Induction

AbstractThere is substantial interest in memory reconsolidation as a target for the treatment of anxiety disorders such as post-traumatic stress disorder (PTSD). However, its applicability is restricted by reconsolidation-resistant conditions that constrain the initial memory destabilization. In this study, we investigated whether the induction of synaptic protein degradation through autophagy modulation, a major protein degradation pathway, can enhance memory destabilization upon retrieval and whether it can be utilized to overcome these conditions. Here, using male mice in an auditory fear reconsolidation model, we showed that autophagy contributes to memory destabilization and its induction can be utilized to enhance erasure of a reconsolidation-resistant auditory fear memory that depended on α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptor (AMPAR) endocytosis. Using male mice in a contextual fear reconsolidation model, autophagy induction in the amygdala or in the hippocampus enhanced fear or contextual memory destabilization, respectively. The latter correlated with AMPAR degradation in the spines of the contextual memory-ensemble cells. Using male rats in an in vivo long-term potentiation reconsolidation model, autophagy induction enhanced synaptic destabilization in an N-methyl-D-aspartate receptor-dependent manner. These data indicate that induction of synaptic protein degradation can enhance both synaptic and memory destabilization upon reactivation and that autophagy inducers have the potential to be used as a therapeutic tool in the treatment of anxiety disorders.Significance StatementIt has been reported that inhibiting synaptic protein degradation prevents memory destabilization. However, whether the reverse relation is true and whether it can be utilized to enhance memory destabilization is still unknown. Here we addressed this question on the behavioral, molecular and synaptic levels, and showed that induction of autophagy, a major protein degradation pathway, can enhance memory and synaptic destabilization upon reactivation. We also show that autophagy induction can be utilized to overcome a reconsolidation-resistant memory, suggesting autophagy inducers as a potential therapeutic tool in the treatment of anxiety disorders.

Abstract P229: cGMP Stimulates Ubiquitination and Degradation of NKCC2 in Thick Ascending Limbs

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Gustavo R Ares
Keyword(s):  
Protein Degradation ◽  
Apical Membrane ◽  
Proteasome Inhibition ◽  
Ubiquitin Proteasome System ◽  
Surface Proteins ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
General Protein

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter (NKCC2). Nitric oxide inhibits NaCl reabsorption in TALs by increasing the second messenger cyclic guanylate monophosphate (cGMP) and decreasing NKCC2 in the apical plasma membrane. Recently, we showed that internalized NKCC2 is constitutively degraded (0.33%/min). Protein degradation regulates channels and transporters activity along the nephron. However, whether NKCC2 is regulated by lysosomal degradation or ubiquitin-proteasomal system remains unknown. We hypothesized that internalized NKCC2 is degraded by the ubiquitin-proteasome system in a process stimulated by cGMP. TAL surface proteins were biotinylated and allowed to internalize. The biotin remaining in surface proteins was stripped away and only internalized NKCC2 measured by Western blot. cGMP enhanced the rate of disappearance of internalized NKCC2 by 83 % and this was blocked by a proteasomal (MG132) but not lysosomal (leupeptin) inhibitor (Control: 0.29 ± 0.04; cGMP: 0.53 ± 0.10 ; cGMP + MG132: 0.10 ± 0.10 ; cGMP + Leupeptin: 0.44 ± 0.06 %/min; p < 0.05). In general, protein degradation by the proteasomal system requires ubiquitination of the targeted protein. We found that NKCC2 inmuno-precipitated with ubiquitin. Proteasome inhibition induced the accumulation of ubiquitin-NKCC2 and this was enhanced by cGMP (MG132: 59 ± 14%, MG132+cGMP: 111 ± 25%; p <0.05). We concluded that internalized NKCC2 is degraded via the ubiquitin-proteasome pathway in a process stimulated by cGMP. cGMP-induced degradation of internalized NKCC2 may contribute to decreased NKCC2 trafficking to the apical membrane therefore decreasing NaCl reabsorption in TALs.

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