scholarly journals SUFFICIENT ACTIVITY OF THE UBIQUITIN PROTEASOME SYSTEM IN AGED MICE AND DURING RETINAL DEGENERATION SUPPORTS DHFR-BASED CONDITIONAL CONTROL OF PROTEIN ABUNDANCE IN THE RETINA

2021 ◽  
Author(s):  
Hui Peng ◽  
Prerana Ramadurgum ◽  
DaNae R. Woodard ◽  
Steffi Daniel ◽  
Marian Renwick ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Mouse Models ◽  
Ubiquitin Proteasome System ◽  
Model Systems ◽  
Protein Abundance ◽  
Aged Mice ◽  
Gene Therapies ◽  
Conditional Control ◽  
Ubiquitin Proteasome ◽  
Regulate Protein

SummaryThe Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. In the absence of TMP, a DHFR stabilizer, the DD is degraded by the ubiquitin proteasome system (UPS). To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, which may have a compromised UPS, we evaluated the DHFR DD system in aged mice (up to 24 mo), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4−/− mice). Aged, LIRD, and Abca4−/− mice all had similar proteasomal activities and high-molecular weight ubiquitin levels compared to control mice. However, rd2 mice displayed compromised chymotrypsin activity compared to control mice. Nonetheless, the DHFR DD was effectively degraded in all model systems, including rd2 mice. Moreover, TMP increased DHFR DD-dependent retinal bioluminescence in all mouse models, however the fold induction was slightly, albeit significantly, lower in Abca4−/− mice. Thus, the destabilized DHFR DD-based approach allows for efficient control of protein abundance in aged mice and retinal degeneration mouse models, laying the foundation to use this strategy in a wide variety of mice for the conditional control of gene therapies to potentially treat multiple eye diseases.

scholarly journals Molecular Chaperones and Proteolytic Machineries Regulate Protein Homeostasis in Aging Cells

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1308 ◽  
Author(s):  
Boris Margulis ◽  
Anna Tsimokha ◽  
Svetlana Zubova ◽  
Irina Guzhova
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Molecular Chaperones ◽  
Ubiquitin Proteasome System ◽  
Life Cycles ◽  
Protein Homeostasis ◽  
Ubiquitin Proteasome ◽  
Correct Function ◽  
Regulate Protein

Throughout their life cycles, cells are subject to a variety of stresses that lead to a compromise between cell death and survival. Survival is partially provided by the cell proteostasis network, which consists of molecular chaperones, a ubiquitin-proteasome system of degradation and autophagy. The cooperation of these systems impacts the correct function of protein synthesis/modification/transport machinery starting from the adaption of nascent polypeptides to cellular overcrowding until the utilization of damaged or needless proteins. Eventually, aging cells, in parallel to the accumulation of flawed proteins, gradually lose their proteostasis mechanisms, and this loss leads to the degeneration of large cellular masses and to number of age-associated pathologies and ultimately death. In this review, we describe the function of proteostasis mechanisms with an emphasis on the possible associations between them.

scholarly journals Systematic approaches to identify E3 ligase substrates

2016 ◽  
Vol 473 (22) ◽  
pp. 4083-4101 ◽  
Author(s):  
Mary Iconomou ◽  
Darren N. Saunders
Keyword(s):  
Cell Cycle Progression ◽  
Target Identification ◽  
Ubiquitin Proteasome System ◽  
Model Systems ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Diverse Range ◽  
E3 Ligases ◽  
Ubiquitin Proteasome ◽  
Substrate Proteins

Protein ubiquitylation is a widespread post-translational modification, regulating cellular signalling with many outcomes, such as protein degradation, endocytosis, cell cycle progression, DNA repair and transcription. E3 ligases are a critical component of the ubiquitin proteasome system (UPS), determining the substrate specificity of the cascade by the covalent attachment of ubiquitin to substrate proteins. Currently, there are over 600 putative E3 ligases, but many are poorly characterized, particularly with respect to individual protein substrates. Here, we highlight systematic approaches to identify and validate UPS targets and discuss how they are underpinning rapid advances in our understanding of the biochemistry and biology of the UPS. The integration of novel tools, model systems and methods for target identification is driving significant interest in drug development, targeting various aspects of UPS function and advancing the understanding of a diverse range of disease processes.

scholarly journals Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits

2011 ◽  
Vol 286 (22) ◽  
pp. 19191-19203 ◽  
Author(s):  
Makoto Miyazawa ◽  
Erika Tashiro ◽  
Hirotake Kitaura ◽  
Hiroshi Maita ◽  
Hiroo Suto ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Proteasome Inhibitor ◽  
Binding Protein ◽  
Ubiquitin Proteasome System ◽  
Unfolded Proteins ◽  
Protein Levels ◽  
Mutual Regulation ◽  
Ubiquitin Proteasome ◽  
Transformation Activity ◽  
Regulate Protein

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

scholarly journals The proteasome activity reporter GFP-Cl1 is degraded by autophagy in the aging model Podospora anserina

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 230 ◽  
Author(s):  
Matthias Wiemer ◽  
Heinz D. Osiewacz
Keyword(s):  
Protein Quality ◽  
Ubiquitin Proteasome System ◽  
Substantial Effect ◽  
Podospora Anserina ◽  
Proteasome Activity ◽  
Protein Abundance ◽  
Wild Type ◽  
Vital Function ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome

The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina.

Transgenic manipulation of the ubiquitin–proteasome system

2005 ◽  
Vol 41 ◽  
pp. 129-138
Author(s):  
Douglas A. Gray
Keyword(s):  
Mouse Model ◽  
Basic Research ◽  
Ubiquitin Proteasome System ◽  
Industrial Applications ◽  
Model Systems ◽  
Transgenic Organisms ◽  
Plant Nematode ◽  
Ubiquitin Proteasome ◽  
Research Questions ◽  
Transgenic Manipulation

The transgenic approach has been used in model systems from yeast to mammals to address basic research questions, and to achieve agricultural, pharmaceutical or industrial objectives. In basic research, transgenic organisms have generated novel observations that could not have been obtained otherwise. This chapter concentrates on the use of transgenics in deciphering the operation of the UPS (ubiquitin–proteasome system) in the yeast, plant, nematode, fly, and mouse model systems, and will touch on ways in which transgenic manipulation of the UPS has been exploited for agricultural, pharmaceutical, and industrial applications.

scholarly journals The Role of Autophagy in Chemical Proteasome Inhibition Model of Retinal Degeneration

2021 ◽  
Vol 22 (14) ◽  
pp. 7271
Author(s):  
Merry Gunawan ◽  
Choonbing Low ◽  
Kurt Neo ◽  
Siawey Yeo ◽  
Candice Ho ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Structural Integrity ◽  
Mammalian Target Of Rapamycin ◽  
Neuroblastoma Cells ◽  
Proteasome Inhibition ◽  
Ubiquitin Proteasome System ◽  
Ocular Tissue ◽  
Protective Mechanism ◽  
Ubiquitin Proteasome

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin–proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.

scholarly journals The JAK2-STAT3 pathway controls a beneficial proteostasis response of reactive astrocytes in Huntington’s disease

2021 ◽  
Author(s):  
L. Abjean ◽  
L. Ben Haim ◽  
M. Riquelme-Perez ◽  
P. Gipchtein ◽  
C. Derbois ◽  
...  
Keyword(s):  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Mouse Models ◽  
Ubiquitin Proteasome System ◽  
Reactive Astrocytes ◽  
Stat3 Pathway ◽  
Functional Changes ◽  
Ubiquitin Proteasome ◽  
Open Question ◽  
Proteolytic Capacity

AbstractHuntington’s disease (HD) is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin (mHTT) and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in HD that involves morphological, molecular and functional changes. How reactive astrocytes contribute to HD is still an open question, especially because their reactive state is poorly reproduced in mouse models.Here, we show that the JAK2-STAT3 pathway, a central cascade controlling the reactive response of astrocytes, is activated in the putamen of HD patients. Selective activation of this cascade in astrocytes reduces the number and size of neuronal mHTT aggregates and improves neuronal features in two HD mouse models. Moreover, activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomes and the ubiquitin-proteasome system, and enhances their production of the co-chaperone DNAJB1, which is released in exosomes.Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in HD, which involves bi-directional signalling with neurons to reduce mHTT aggregation and toxicity.

scholarly journals The USP28-ΔNp63 axis is a vulnerability of squamous tumours

2019 ◽  
Author(s):  
Cristian Prieto-Garcia ◽  
Oliver Hartmann ◽  
Michaela Reissland ◽  
Fabian Braun ◽  
Thomas Fischer ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Therapeutic Strategy ◽  
Ubiquitin Proteasome System ◽  
Lung Tumour ◽  
Protein Abundance ◽  
Growth And Survival ◽  
Murine Lung ◽  
Ubiquitin Proteasome

AbstractThe transcription factor ΔNp63 is a master regulator that establishes epithelial cell identity and is essential for the survival of SCC of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ΔNp63 protein and maintains elevated ΔNP63 levels in SCC by counteracting its proteasome-mediated degradation. Interference with USP28 activity by genetic means abolishes the transcriptional identity of SCC cells and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered mouse models establish that both induction and maintenance of lung SCC strictly depend on endogenous USP28. Targeting ΔNp63 protein abundance in SCC via inhibition of USP28 therefore is a feasible strategy for the treatment of SCC tumours.SignificanceSCC depend on ΔNp63, and its protein abundance is tightly controlled by the ubiquitin proteasome system. Here, we demonstrate the dependence of SCC on USP28 for various human SCC in vitro and in vivo using murine lung tumour models. As inhibitors for deubiquitylases become available, targeting USP28 is a promising therapeutic strategy.

The aggravating role of the ubiquitin–proteasome system in neurodegenerative disease

2006 ◽  
Vol 34 (5) ◽  
pp. 743-745 ◽  
Author(s):  
C.-C. Hung ◽  
E.J. Davison ◽  
P.A. Robinson ◽  
H.C. Ardley
Keyword(s):  
Ubiquitin Proteasome System ◽  
Model Systems ◽  
Protective Mechanism ◽  
Ubiquitinated Proteins ◽  
Age Related ◽  
Ubiquitin Proteasome ◽  
Vitro Model ◽  
Novel Therapeutic Strategies

Intraneuronal inclusion bodies are key pathological features of most age-related neurodegenerative disorders including Parkinson's disease and Alzheimer's disease. These inclusions are commonly characterized both by the presence of ubiquitinated proteins and the sequestration of components of the UPS (ubiquitin–proteasome system). Unfortunately, as we age, the efficiency of the UPS declines, suggesting that the presence of ubiquitinated proteins and UPS components in inclusions may reflect unsuccessful attempts by the (failing) UPS to remove the aggregating proteins. Whether the physical presence of inclusions causes cell death or, conversely, whether they are non-toxic and their presence reflects a cellular protective mechanism remains highly controversial. Animal and in vitro model systems that allow detailed characterization of the inclusions and their effects on the cell have been developed by us and others. Identification of the mechanisms involved in inclusion formation is already aiding the development of novel therapeutic strategies to prevent or alleviate aggregate-associated neurodegenerative diseases.

The ubiquitin–proteasome system and skeletal muscle wasting

2005 ◽  
Vol 41 ◽  
pp. 173-186 ◽  
Author(s):  
Didier Attaix ◽  
Sophie Ventadour ◽  
Audrey Codran ◽  
Daniel Béchet ◽  
Daniel Taillandier ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Wasting ◽  
Proteolytic Enzymes ◽  
Cathepsin L ◽  
Ubiquitin Proteasome System ◽  
Complete Breakdown ◽  
Skeletal Muscle Proteins ◽  
Ubiquitin Proteasome ◽  
Tripeptidyl Peptidase Ii ◽  
F Box Protein

The ubiquitin–proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin–protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

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