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The Journal of Cell Biology Sep 2023Glutathionylation is a posttranslational modification involved in various molecular and cellular processes. However, it remains unknown whether and how glutathionylation...
Glutathionylation is a posttranslational modification involved in various molecular and cellular processes. However, it remains unknown whether and how glutathionylation regulates nervous system development. To identify critical regulators of synapse growth and development, we performed an RNAi screen and found that postsynaptic knockdown of glutathione transferase omega 1 (GstO1) caused significantly more synaptic boutons at the Drosophila neuromuscular junctions. Genetic and biochemical analysis revealed an increased level of glass boat bottom (Gbb), the Drosophila homolog of mammalian bone morphogenetic protein (BMP), in GstO1 mutants. Further experiments showed that GstO1 is a critical regulator of Gbb glutathionylation at cysteines 354 and 420, which promoted its degradation via the proteasome pathway. Moreover, the E3 ligase Ctrip negatively regulated the Gbb protein level by preferentially binding to glutathionylated Gbb. These results unveil a novel regulatory mechanism in which glutathionylation of Gbb facilitates its ubiquitin-mediated degradation. Taken together, our findings shed new light on the crosstalk between glutathionylation and ubiquitination of Gbb in synapse development.
Topics: Animals; Drosophila; Neuromuscular Junction; Proteasome Endopeptidase Complex; Synapses; Drosophila Proteins
PubMed: 37389657
DOI: 10.1083/jcb.202202068 -
Cells Dec 2023Ubiquitination is a reversible post-translational modification based on the chemical addition of ubiquitin to proteins with regulatory effects on various signaling... (Review)
Review
Ubiquitination is a reversible post-translational modification based on the chemical addition of ubiquitin to proteins with regulatory effects on various signaling pathways. Ubiquitination can alter the molecular functions of tagged substrates with respect to protein turnover, biological activity, subcellular localization or protein-protein interaction. As a result, a wide variety of cellular processes are under ubiquitination-mediated control, contributing to the maintenance of cellular homeostasis. It follows that the dysregulation of ubiquitination reactions plays a relevant role in the pathogenic states of human diseases such as neurodegenerative diseases, immune-related pathologies and cancer. In recent decades, the enzymes of the ubiquitin-proteasome system (UPS), including E3 ubiquitin ligases and deubiquitinases (DUBs), have attracted attention as novel druggable targets for the development of new anticancer therapeutic approaches. This perspective article summarizes the peculiarities shared by the enzymes involved in the ubiquitination reaction which, when deregulated, can lead to tumorigenesis. Accordingly, an overview of the main pharmacological interventions based on targeting the UPS that are in clinical use or still in clinical trials is provided, also highlighting the limitations of the therapeutic efficacy of these approaches. Therefore, various attempts to circumvent drug resistance and side effects as well as UPS-related emerging technologies in anticancer therapeutics are discussed.
Topics: Humans; Cytoplasm; Neoplasms; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Ubiquitin; Ubiquitination
PubMed: 38201233
DOI: 10.3390/cells13010029 -
The Journal of Cell Biology Jun 2024Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription...
Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.
Topics: Animals; Humans; Mice; Autophagy; Lysosomes; NF-E2-Related Factor 1; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteostasis; Proteotoxic Stress; Stress, Physiological
PubMed: 38656405
DOI: 10.1083/jcb.202306150 -
Cancer Letters Jul 2024Ubiquitination and related cellular processes control a variety of aspects in human cell biology, and defects in these processes contribute to multiple illnesses. In... (Review)
Review
Ubiquitination and related cellular processes control a variety of aspects in human cell biology, and defects in these processes contribute to multiple illnesses. In recent decades, our knowledge about the pathological role of ubiquitination in lymphoid cancers and therapeutic strategies to target the modified ubiquitination system has evolved tremendously. Here we review the altered signalling mechanisms mediated by the aberrant expression of cancer-associated E2s/E3s and deubiquitinating enzymes (DUBs), which result in the hyperactivation of oncoproteins or the frequently allied downregulation of tumour suppressors. We discuss recent highlights pertaining to the several different therapeutic interventions which are currently being evaluated to effectively block abnormal ubiquitin-proteasome pathway and the use of heterobifunctional molecules which recruit the ubiquitination system to degrade or stabilize non-cognate substrates. This review aids in comprehension of ubiquitination aberrance in lymphoid cancers and current targeting strategies and elicits further investigations to deeply understand the link between cellular ubiquitination and lymphoid pathogenesis as well as to ameliorate corresponding treatment interventions.
Topics: Humans; Ubiquitination; Signal Transduction; Ubiquitin; Animals; Lymphoma; Molecular Targeted Therapy; Antineoplastic Agents; Proteasome Endopeptidase Complex; Deubiquitinating Enzymes
PubMed: 38795760
DOI: 10.1016/j.canlet.2024.216978 -
Viruses Sep 2023Although the involvement of the ubiquitin-proteasome system (UPS) in several coronavirus-productive infections has been reported, whether the UPS is required for...
Although the involvement of the ubiquitin-proteasome system (UPS) in several coronavirus-productive infections has been reported, whether the UPS is required for infectious bronchitis virus (IBV) and porcine epidemic diarrhea virus (PEDV) infections is unclear. In this study, the role of UPS in the IBV and PEDV life cycles was investigated. When the UPS was suppressed by pharmacological inhibition at the early infection stage, IBV and PEDV infectivity were severely impaired. Further study showed that inhibition of UPS did not change the internalization of virus particles; however, by using R18 and DiOC-labeled virus particles, we found that inhibition of UPS prevented the IBV and PEDV membrane fusion with late endosomes or lysosomes. In addition, proteasome inhibitors blocked the degradation of the incoming viral protein N, suggesting the uncoating process and genomic RNA release were suppressed. Subsequently, the initial translation of genomic RNA was blocked. Thus, UPS may target the virus-cellular membrane fusion to facilitate the release of incoming viruses from late endosomes or lysosomes, subsequently blocking the following virus uncoating, initial translation, and replication events. Similar to the observation of proteasome inhibitors, ubiquitin-activating enzyme E1 inhibitor PYR-41 also impaired the entry of IBV, enhanced the accumulation of ubiquitinated proteins, and depleted mono-ubiquitin. In all, this study reveals an important role of UPS in coronavirus entry by preventing membrane fusion and identifies UPS as a potential target for developing antiviral therapies for coronavirus.
Topics: Animals; Swine; Proteasome Endopeptidase Complex; Cell Line; Ubiquitin; Coronavirus; Proteasome Inhibitors; Membrane Fusion; Coronavirus Infections; Endosomes; Porcine epidemic diarrhea virus; RNA; Virus Replication
PubMed: 37896778
DOI: 10.3390/v15102001 -
Cell Death & Disease Aug 2023Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein...
Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. In this study, we found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. Our findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process.
Topics: Animals; Autophagy; Autophagosomes; Cell Death; Drosophila; Lysosomes; Proteasome Endopeptidase Complex; Ubiquitin; Deubiquitinating Enzymes; Autophagy-Related Protein-1 Homolog; Drosophila Proteins; Ubiquitin-Specific Proteases
PubMed: 37607937
DOI: 10.1038/s41419-023-06062-x -
Molecules (Basel, Switzerland) Sep 2023Ubiquitin, a small protein, is well known for tagging target proteins through a cascade of enzymatic reactions that lead to protein degradation. The ubiquitin tag, apart... (Review)
Review
Ubiquitin, a small protein, is well known for tagging target proteins through a cascade of enzymatic reactions that lead to protein degradation. The ubiquitin tag, apart from its signaling role, is paramount in destabilizing the modified protein. Here, we explore the complex role of ubiquitin-mediated protein destabilization in the intricate proteolysis process by the 26S proteasome. In addition, the significance of the so-called ubiquitin-independent pathway and the role of the 20S proteasome are considered. Next, we discuss the ubiquitin-proteasome system's interplay with pathogenic microorganisms and how the microorganisms manipulate this system to establish infection by a range of elaborate pathways to evade or counteract host responses. Finally, we focus on the mechanisms that rely either on (i) hijacking the host and on delivering pathogenic E3 ligases and deubiquitinases that promote the degradation of host proteins, or (ii) counteracting host responses through the stabilization of pathogenic effector proteins.
PubMed: 37764516
DOI: 10.3390/molecules28186740 -
Neoplasia (New York, N.Y.) Sep 2023The Sonic Hedgehog (Hh) signal transduction pathway plays a critical role in many developmental processes and, when deregulated, may contribute to several cancers,...
The Sonic Hedgehog (Hh) signal transduction pathway plays a critical role in many developmental processes and, when deregulated, may contribute to several cancers, including basal cell carcinoma, medulloblastoma, colorectal, prostate, and pancreatic cancer. In recent years, several Hh inhibitors have been developed, mainly acting on the Smo receptor. However, drug resistance due to Smo mutations or non-canonical Hh pathway activation highlights the need to identify further mechanisms of Hh pathway modulation. Among these, deacetylation of the Hh transcription factor Gli1 by the histone deacetylase HDAC1 increases Hh activity. On the other end, the KCASH family of oncosuppressors binds HDAC1, leading to its ubiquitination and subsequent proteasomal degradation, leaving Gli1 acetylated and not active. It was recently demonstrated that the potassium channel containing protein KCTD15 is able to interact with KCASH2 protein and stabilize it, enhancing its effect on HDAC1 and Hh pathway. KCTD15 and KCTD1 proteins share a high homology and are clustered in a specific KCTD subfamily. We characterize here KCTD1 role on the Hh pathway. Therefore, we demonstrated KCTD1 interaction with KCASH1 and KCASH2 proteins, and its role in their stabilization by reducing their ubiquitination and proteasome-mediated degradation. Consequently, KCTD1 expression reduces HDAC1 protein levels and Hh/Gli1 activity, inhibiting Hh dependent cell proliferation in Hh tumour cells. Furthermore, analysis of expression data on publicly available databases indicates that KCTD1 expression is reduced in Hh dependent MB samples, compared to normal cerebella, suggesting that KCTD1 may represent a new putative target for therapeutic approaches against Hh-dependent tumour.
Topics: Male; Humans; Hedgehog Proteins; Zinc Finger Protein GLI1; Cell Proliferation; Databases, Factual; Cerebellar Neoplasms; Co-Repressor Proteins
PubMed: 37597490
DOI: 10.1016/j.neo.2023.100926 -
Journal of Medicinal Chemistry Aug 2023There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the...
There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the proteasome. A related analogue, active against , showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.
Topics: Mice; Animals; Trypanosoma cruzi; Proteasome Inhibitors; Proteasome Endopeptidase Complex; Chagas Disease; Leishmaniasis, Visceral; Trypanocidal Agents
PubMed: 37506194
DOI: 10.1021/acs.jmedchem.3c00582 -
Nature Communications Sep 2023In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting...
In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation.
Topics: Autophagy; Autophagosomes; Cytosol; Macroautophagy; Ribosomes
PubMed: 37726301
DOI: 10.1038/s41467-023-41525-x