Negative Regulator of Ubiquitin-Like Protein 1 modulates the autophagy–lysosomal pathway via p62 to facilitate the extracellular release of tau following proteasome impairment

Abstract Negative regulator of ubiquitin-like protein 1 (NUB1) and its longer isoform NUB1L are ubiquitin-like (UBL)/ubiquitin-associated (UBA) proteins that facilitate the targeting of proteasomal substrates, including tau, synphilin-1 and huntingtin. Previous data revealed that NUB1 also mediated a reduction in tau phosphorylation and aggregation following proteasome inhibition, suggesting a switch in NUB1 function from targeted proteasomal degradation to a role in autophagy. Here, we delineate the mechanisms of this switch and show that NUB1 interacted specifically with p62 and induced an increase in p62 levels in a manner facilitated by inhibition of the proteasome. NUB1 moreover increased autophagosomes and the recruitment of lysosomes to aggresomes following proteasome inhibition. Autophagy flux assays revealed that NUB1 affected the autophagy–lysosomal pathway primarily via the UBA domain. NUB1 localized to cytosolic inclusions with pathological forms of tau, as well as LAMP1 and p62 in the hippocampal neurons of tauopathy mice. Finally, NUB1 facilitated the extracellular release of tau following proteasome inhibition. This study thus shows that NUB1 plays a role in regulating the autophagy–lysosomal pathway when the ubiquitin proteasome system is compromised, thus contributing to the mechanisms targeting the removal of aggregation-prone proteins upon proteasomal impairment.

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Immunoproteasome Function in Normal and Malignant Hematopoiesis

Cells ◽

10.3390/cells10071577 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1577

Author(s):

Nuria Tubío-Santamaría ◽

Frédéric Ebstein ◽

Florian H. Heidel ◽

Elke Krüger

Keyword(s):

Proteasome Inhibition ◽

Ubiquitin Proteasome System ◽

Hematologic Malignancies ◽

Protein Homeostasis ◽

Efficacy And Safety ◽

Hematopoietic System ◽

Oxidized Proteins ◽

Attractive Therapeutic Target ◽

Ubiquitin Proteasome ◽

Early Phases

The ubiquitin–proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.

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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.

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USP7 inhibits Wnt/β-catenin signaling through promoting stabilization of Axin

Nature Communications ◽

10.1038/s41467-019-12143-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 14

Author(s):

Lei Ji ◽

Bo Lu ◽

Raffaella Zamponi ◽

Olga Charlat ◽

Robert Aversa ◽

...

Keyword(s):

Osteoblast Differentiation ◽

Adipocyte Differentiation ◽

Ubiquitin Proteasome System ◽

Cellular Systems ◽

Negative Regulator ◽

Scaffolding Protein ◽

Complex Stability ◽

Genetic Ablation ◽

Ubiquitin Proteasome ◽

Potent Negative Regulator

Abstract Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.

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The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

The Journal of Cell Biology ◽

10.1083/jcb.201509039 ◽

2016 ◽

Vol 212 (7) ◽

pp. 789-801 ◽

Cited By ~ 20

Author(s):

Maria J. Pinto ◽

Pedro L. Alves ◽

Luís Martins ◽

Joana R. Pedro ◽

Hyun R. Ryu ◽

...

Keyword(s):

Synaptic Vesicles ◽

Proteasome Inhibition ◽

Ubiquitin Proteasome System ◽

Presynaptic Terminal ◽

Ubiquitinated Proteins ◽

Presynaptic Differentiation ◽

Intrinsic Mechanism ◽

Ubiquitin Proteasome ◽

Presynaptic Assembly ◽

Local Modulation

Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin–proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses.

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p97 Negatively Regulates NRF2 by Extracting Ubiquitylated NRF2 from the KEAP1-CUL3 E3 Complex

Molecular and Cellular Biology ◽

10.1128/mcb.00660-16 ◽

2017 ◽

Vol 37 (8) ◽

Cited By ~ 36

Author(s):

Shasha Tao ◽

Pengfei Liu ◽

Gang Luo ◽

Montserrat Rojo de la Vega ◽

Heping Chen ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Protein Complexes ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Ubiquitin Ligase Complex ◽

Ubiquitin Proteasome ◽

Client Proteins

ABSTRACT Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation (the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system (UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX-containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.

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Gfi1 ubiquitination and proteasomal degradation is inhibited by the ubiquitin ligase Triad1

Blood ◽

10.1182/blood-2006-11-058602 ◽

2007 ◽

Vol 110 (9) ◽

pp. 3128-3135 ◽

Cited By ~ 22

Author(s):

Jurgen A. F. Marteijn ◽

Laurens T. van der Meer ◽

Liesbeth van Emst ◽

Simon van Reijmersdal ◽

Willemijn Wissink ◽

...

Keyword(s):

Cell Proliferation ◽

Ubiquitin Ligase ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Binding Domain ◽

U937 Cells ◽

Dna Binding Domain ◽

Protein Levels ◽

Ubiquitin Ligase Activity ◽

Ubiquitin Proteasome

Abstract Growth factor independence 1 (Gfi1) is a transcriptional repressor essential for the function and development of many different hematopoietic lineages. The Gfi1 protein expression is regulated by the ubiquitin-proteasome system. In granulocytes, Gfi1 is rapidly degraded by the proteasome, while it is more stable in monocytes. How the ubiquitination and degradation of Gfi1 is regulated is unclear. Here, we show that the ubiquitin ligase Triad1 interacts with the DNA-binding domain of Gfi1. Unexpectedly, we found that Triad1 inhibited Gfi1 ubiquitination, resulting in a prolonged half-life. Down-regulation of endogenous Triad1 by siRNAs resulted in increased Gfi1 ubiquitination. In U937 cells, Triad1 caused an increase in endogenous Gfi1 protein levels and slowed cell proliferation in a similar manner when Gfi1 itself was expressed. A Triad1 mutant that lacks the Gfi1-binding domain did not affect Gfi1 levels and proliferation. Because neither proteasome-ubiquitin nor Triad1 ubiquitin ligase activity was required for the inhibition of Gfi1 ubiquitination, these data suggest that Triad1 competes for Gfi1 binding with as yet to be identified E3 ubiquitin ligases that do mark Gfi1 for proteasomal degradation. The finetuning of Gfi1 protein levels regulated by Triad1 defines an unexpected role for this protein in hematopoiesis.

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The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans

The Journal of Cell Biology ◽

10.1083/jcb.200607084 ◽

2007 ◽

Vol 177 (2) ◽

pp. 205-210 ◽

Cited By ~ 61

Author(s):

Megan L. Landsverk ◽

Shumin Li ◽

Alex H. Hutagalung ◽

Ayaz Najafov ◽

Thorsten Hoppe ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Proteasome Inhibition ◽

Thick Filament ◽

Ubiquitin Proteasome System ◽

Loss Of Function ◽

Cellular Processes ◽

Ubiquitin Proteasome ◽

Myosin Motors ◽

And Function ◽

Sarcomere Assembly

Myosin motors are central to diverse cellular processes in eukaryotes. Homologues of the myosin chaperone UNC-45 have been implicated in the assembly and function of myosin-containing structures in organisms from fungi to humans. In muscle, the assembly of sarcomeric myosin is regulated to produce stable, uniform thick filaments. Loss-of-function mutations in Caenorhabditis elegans UNC-45 lead to decreased muscle myosin accumulation and defective thick filament assembly, resulting in paralyzed animals. We report that transgenic worms overexpressing UNC-45 also display defects in myosin assembly, with decreased myosin content and a mild paralysis phenotype. We find that the reduced myosin accumulation is the result of degradation through the ubiquitin/proteasome system. Partial proteasome inhibition is able to restore myosin protein and worm motility to nearly wild-type levels. These findings suggest a mechanism in which UNC-45–related proteins may contribute to the degradation of myosin in conditions such as heart failure and muscle wasting.

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Diminished proteasomal degradation results in accumulation of Gfi1 protein in monocytes

Blood ◽

10.1182/blood-2006-02-003590 ◽

2006 ◽

Vol 109 (1) ◽

pp. 100-108 ◽

Cited By ~ 14

Author(s):

Jurgen A. F. Marteijn ◽

Laurens T. van der Meer ◽

Liesbeth Van Emst ◽

Theo de Witte ◽

Joop H. Jansen ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Myeloid Differentiation ◽

Mrna Levels ◽

Monocytic Differentiation ◽

Granulocytic Differentiation ◽

Monocytic Cell ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Monocytic Lineage

Abstract Gfi1 is a transcriptional repressor essential during myeloid differentiation. Gfi1−/− mice exhibit a block in myeloid differentiation resulting in the accumulation of an immature myelo-monocytic cell population and the complete absence of mature neutrophils. Even though mRNA levels of Gfi1 appear to be very low in monocytes, Gfi1 might play a role in the monocytic lineage as Gfi1−/− mice exhibit diminished monocyte-derived dendritic cells and disturbed cytokine production by macrophages in response to LPS. We show here that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Upon forced monocytic differentiation of U937 cells, Gfi1 mRNA levels dropped but protein levels increased due to diminished proteasomal turnover. Similarly, Gfi1 mRNA levels are low in primary monocytes whereas the protein is clearly detectable. Conversely, Gfi1 mRNA levels are high in granulocytes but the protein is swiftly degraded by the proteasome in these cells. Chromatin immunoprecipitation experiments showed that Gfi1 binds to the promoter of several granulocyte-specific genes in primary monocytes, including C/EBPα, neutrophil elastase, and Gfi1 itself. The binding of the repressor Gfi1 to these promoters correlated with low expression of these genes in monocytes compared with granulocytes. Our data fit a model in which Gfi1 protein levels are induced in primary monocytes, due to diminished proteasomal degradation, to repress genes that play a role in granulocytic differentiation.

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The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover

Molecular Biology of the Cell ◽

10.1091/mbc.e10-09-0748 ◽

2011 ◽

Vol 22 (3) ◽

pp. 291-300 ◽

Cited By ~ 158

Author(s):

Shan Xu ◽

Guihong Peng ◽

Yang Wang ◽

Shengyun Fang ◽

Mariusz Karbowski

Keyword(s):

Mitochondrial Membrane ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Degradation Pathway ◽

Outer Mitochondrial Membrane ◽

Aaa Atpase ◽

Associated Proteins ◽

Ubiquitin Proteasome ◽

Molecular Components

Recent studies have revealed a role for the ubiquitin/proteasome system in the regulation and turnover of outer mitochondrial membrane (OMM)-associated proteins. Although several molecular components required for this process have been identified, the mechanism of proteasome-dependent degradation of OMM-associated proteins is currently unclear. We show that an AAA-ATPase, p97, is required for the proteasomal degradation of Mcl1 and Mfn1, two unrelated OMM proteins with short half-lives. A number of biochemical assays, as well as imaging of changes in localization of photoactivable GFP-fused Mcl1, revealed that p97 regulates the retrotranslocation of Mcl1 from mitochondria to the cytosol, prior to, or concurrent with, proteasomal degradation. Mcl1 retrotranslocation from the OMM depends on the activity of the ATPase domain of p97. Furthermore, p97-mediated retrotranslocation of Mcl1 can be recapitulated in vitro, confirming a direct mitochondrial role for p97. Our results establish p97 as a novel and essential component of the OMM-associated protein degradation pathway.

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