The ubiquitin-proteasome system (UPS) plays an important role in the cellular processes for protein quality control and homeostasis. Dysregulation of the UPS has been implicated in numerous diseases, including cancer. Indeed, components of UPS are frequently mutated or abnormally expressed in various cancers. Since Bortezomib, a proteasome inhibitor, received FDA approval for the treatment of multiple myeloma and mantle cell lymphoma, increasing numbers of researchers have been seeking drugs targeting the UPS as a cancer therapeutic strategy. Here, we introduce the essential component of UPS, including ubiquitinating enzymes, deubiquitinating enzymes and 26S proteasome, and we summarize their targets and mechanisms that are crucial for tumorigenesis. In addition, we briefly discuss some UPS inhibitors, which are currently in clinical trials as cancer therapeutics.

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Ubiquitin–proteasome system (UPS) as a target for anticancer treatment

Protein- and RNA-containing foci and aggregates are a hallmark of many age- and mutation-related neurodegenerative diseases. This article focuses on the role the nucleolus has as a hub in macromolecule regulation in the mammalian nucleus. The nucleolus has a well-established role in ribosome biogenesis and functions in several types of cellular stress responses. In addition to known reactions to DNA damaging and transcription inhibiting stresses, there is an emerging role of the nucleolus especially in responses to proteotoxic stress such as heat shock and inhibition of proteasome function. The nucleolus serves as an active regulatory site for detention of extranucleolar proteins. This takes place in nucleolar cavities and manifests in protein and RNA collections referred to as intranucleolar bodies (INBs), nucleolar aggresomes or amyloid bodies (A-bodies), depending on stress type, severity of accumulation, and material propensities of the macromolecular collections. These indicate a relevance of nucleolar function and regulation in neurodegeneration-related cellular events, but also provide surprising connections with cancer-related pathways. Yet, the molecular mechanisms governing these processes remain largely undefined. In this article, the nucleolus as the site of protein and RNA accumulation and as a possible protective organelle for nuclear proteins during stress is viewed. In addition, recent evidence of liquid-liquid phase separation (LLPS) and liquid-solid phase transition in the formation of nucleoli and its stress responses, respectively, are discussed, along with the increasingly indicated role and open questions for noncoding RNA species in these events.

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Phase-to-Phase With Nucleoli - Stress Responses, Protein Aggregation and Novel Roles of RNA.

Tetramer formation of p53 is essential for its tumor suppressor function. p53 not only acts as a tumor suppressor protein by inducing cell cycle arrest and apoptosis in response to genotoxic stress, but it also regulates other cellular processes, including autophagy, stem cell self-renewal, and reprogramming of differentiated cells into stem cells, immune system, and metastasis. More than 50% of human tumors have TP53 gene mutations, and most of them are missense mutations that presumably reduce tumor suppressor activity of p53. This review focuses on the role of the tetramerization (oligomerization), which is modulated by the protein concentration of p53, posttranslational modifications, and/or interactions with its binding proteins, in regulating the tumor suppressor function of p53. Functional control of p53 by stabilizing or inhibiting oligomer formation and its bio-applications are also discussed. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 598-612, 2016.

Tetramer formation of tumor suppressor protein p53: Structure, function, and applications

Purpose: To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs). Experimental Design: All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs. Results: Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry. Conclusions: These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582–91. ©2016 AACR.

https://clincancerres.aacrjournals.org/content/clincanres/22/22/5582.full.pdf

Significance of TP53 Mutation in Wilms Tumors with Diffuse Anaplasia: A Report from the Children's Oncology Group

Mutations in the oligomerization domain of p53 are genetically linked to cancer susceptibility in Li-Fraumeni Syndrome. These mutations typically alter the oligomeric state of p53 and impair its transcriptional activity. Activation of p53 through tetramerization is required for its tumor suppressive function by inducing transcriptional programs that lead to cell fate decisions such as cell cycle arrest or apoptosis. How p53 chooses between these cell fate outcomes remains unclear. Here, we use 5 oligomeric variants of p53, including 2 novel p53 constructs, that yield either monomeric, dimeric or tetrameric forms of p53 and demonstrate that they induce distinct cellular activities and gene expression profiles that lead to different cell fate outcomes. We report that dimeric p53 variants are cytostatic and can arrest cell growth, but lack the ability to trigger apoptosis in p53-null cells. In contrast, p53 tetramers induce rapid apoptosis and cell growth arrest, while a monomeric variant is functionally inactive, supporting cell growth. In particular, the expression of pro-arrest CDKN1A and pro-apoptotic P53AIP1 genes are important cell fate determinants that are differentially regulated by the oligomeric state of p53. This study suggests that the most abundant oligomeric species of p53 present in resting cells, namely p53 dimers, neither promote cell growth or cell death and that shifting the oligomeric state equilibrium of p53 in cells toward monomers or tetramers is a key parameter in p53-based cell fate decisions.

https://www.tandfonline.com/doi/pdf/10.1080/15384101.2016.1241917

p53 oligomerization status modulates cell fate decisions between growth, arrest and apoptosis.

Spatial management of stress-induced protein aggregation is an integral part of the proteostasis network. Protein modification by the ubiquitin-like molecule NEDD8 increases upon proteotoxic stress and it is characterised by the formation of hybrid NEDD8/ubiquitin conjugates. However, the biological significance of this response is unclear. Combination of quantitative proteomics with biological analysis shows that, during proteotoxic stress, NEDDylation promotes nuclear protein aggregation, including ribosomal proteins as a major group. This correlates with protection of the nuclear Ubiquitin Proteasome System from stress-induced dysfunction. Correspondingly, we show that NEDD8 compromises ubiquitination and prevents targeting and processing of substrates by the proteasome. Moreover, we identify HUWE1 as a key E3-ligase that is specifically required for NEDDylation during proteotoxic stress. The study reveals a specific role for NEDD8 in nuclear protein aggregation upon stress and is consistent with the concept that transient aggregate formation is part of a defence mechanism against proteotoxicity.

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NEDDylation promotes nuclear protein aggregation and protects the Ubiquitin Proteasome System upon proteotoxic stress

https://hal.archives-ouvertes.fr/hal-03176206/document

Proteome-wide identification of NEDD8 modification sites reveals distinct proteomes for canonical and atypical NEDDylation.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.molonc.2014.04.002

Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity

The Ubiquitin-Proteasome System (UPS) plays an important role in the setting of the cellular response to multiple stress signals. Although the primary function of ubiquitin was initially associated with proteolysis, it is now considered as a key regulator of protein function controlling, among other functions, signalling cascades, transcription, apoptosis or oncogenesis. Failure at any level of the UPS is associated with the development of multiple pathologies including metabolic problems, immune diseases, inflammation and cancer. The successful use of the proteasome inhibitor Bortezomib (Velcade) in the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL) revealed the potential of the UPS as pharmacological target. Ten years later, new inhibitors tackling not only the proteasome but also different subsets of enzymes which conjugate or de-conjugate ubiquitin or ubiquitin-like molecules, have been developed. Most of them are excellent tools to characterize better the emerging molecular mechanisms regulating distinct critical cellular processes. Some of them have been launched already while many others are still in pre-clinical development. This chapter updates some of the most successful efforts to develop and characterize inhibitors of the UPS which tackle mechanisms involved in cancer. Particular attention has been dedicated to updating the status of the clinical trials of these inhibitors.

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Sofia Lobato-Gil

Papers

NEDDylation promotes nuclear protein aggregation and protects the Ubiquitin Proteasome System upon proteotoxic stress

Proteome-wide identification of NEDD8 modification sites reveals distinct proteomes for canonical and atypical NEDDylation.

Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity

The Ubiquitin-Proteasome System (UPS) as a Cancer Drug Target: Emerging Mechanisms and Therapeutics