Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment.

The role of cytosolic stress granules in the integrated stress response has remained largely enigmatic. Here, we studied the functionality of the ubiquitin-proteasome system (UPS) in cells that were unable to form stress granules. Surprisingly, the inability of cells to form cytosolic stress granules had primarily a negative impact on the functionality of the nuclear UPS. While defective ribosome products (DRiPs) accumulated at stress granules in thermally stressed control cells, they localized to nucleoli in stress granule-deficient cells. The nuclear localization of DRiPs was accompanied by redistribution and enhanced degradation of SUMOylated proteins. Depletion of the SUMO-targeted ubiquitin ligase RNF4, which targets SUMOylated misfolded proteins for proteasomal degradation, largely restored the functionality of the UPS in the nuclear compartment in stress granule-deficient cells. Stress granule-deficient cells showed an increase in the formation of mutant ataxin-1 nuclear inclusions when exposed to thermal stress. Our data reveal that stress granules play an important role in the sequestration of cytosolic misfolded proteins, thereby preventing these proteins from accumulating in the nucleus, where they would otherwise infringe nuclear proteostasis.

A spider silk-derived solubility domain inhibits nuclear and cytosolic protein aggregation in human cells.

Due to the inherent toxicity of protein aggregates, the propensity of natural, functional amyloidogenic proteins to aggregate must be tightly controlled to avoid negative consequences on cellular viability. The importance of controlled aggregation in biological processes is illustrated by spidroins, which are functional amyloidogenic proteins that form the basis for spider silk. Premature aggregation of spidroins is prevented by the N-terminal NT domain. Here we explored the potential of the engineered, spidroin-based NT* domain in preventing protein aggregation in the intracellular environment of human cells. We show that the NT* domain increases the soluble pool of a reporter protein carrying a ligand-regulatable aggregation domain. Interestingly, the NT* domain prevents the formation of aggregates independent of its position in the aggregation-prone protein. The ability of the NT* domain to inhibit ligand-regulated aggregation was evident both in the cytosolic and nuclear compartments, which are both highly relevant for human disorders linked to non-physiological protein aggregation. We conclude that the spidroin-derived NT* domain has a generic anti-aggregation activity, independent of position or subcellular location, that is also active in human cells and propose that the NT* domain can potentially be exploited in controlling protein aggregation of disease-associated proteins.

Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy.

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.

Reporter-Based Screens for the Ubiquitin/Proteasome System.

Instant and adequate handling of misfolded or otherwise aberrant proteins is of paramount importance for maintaining protein homeostasis in cells. The ubiquitin/proteasome system (UPS) is a central player in protein quality control as it operates in a seek-and-destroy mode, thereby facilitating elimination of faulty proteins. While proteasome inhibition is in clinical use for the treatment of hematopoietic malignancies, stimulation of the UPS has been proposed as a potential therapeutic strategy for various neurodegenerative disorders. High-throughput screens using genetic approaches or compound libraries are powerful tools to identify therapeutic intervention points and novel drugs. Unlike assays that measure specific activities of components of the UPS, reporter substrates provide us with a more holistic view of the general functional status of the UPS in cells. As such, reporter substrates can reveal new ways to obstruct or stimulate this critical proteolytic pathway. Here, we discuss various reporter substrates for the UPS and their application in the identification of key players and the pursuit for novel therapeutics.

The Machado-Joseph disease deubiquitylase ataxin-3 interacts with LC3C/GABARAP and promotes autophagy.

The pathology of spinocerebellar ataxia type 3, also known as Machado-Joseph disease, is triggered by aggregation of toxic ataxin-3 (ATXN3) variants containing expanded polyglutamine repeats. The physiological role of this deubiquitylase, however, remains largely unclear. Our recent work showed that ATX-3, the nematode orthologue of ATXN3, together with the ubiquitin-directed segregase CDC-48, regulates longevity in Caenorhabditis elegans. Here, we demonstrate that the long-lived cdc-48.1; atx-3 double mutant displays reduced viability under prolonged starvation conditions that can be attributed to the loss of catalytically active ATX-3. Reducing the levels of the autophagy protein BEC-1 sensitized worms to the effect of ATX-3 deficiency, suggesting a role of ATX-3 in autophagy. In support of this conclusion, the depletion of ATXN3 in human cells caused a reduction in autophagosomal degradation of proteins. Surprisingly, reduced degradation in ATXN3-depleted cells coincided with an increase in the number of autophagosomes while levels of lipidated LC3 remained unaffected. We identified two conserved LIR domains in the catalytic Josephin domain of ATXN3 that directly interacted with the autophagy adaptors LC3C and GABARAP in vitro. While ATXN3 localized to early autophagosomes, it was not subject to lysosomal degradation, suggesting a transient regulatory interaction early in the autophagic pathway. We propose that the deubiquitylase ATX-3/ATXN3 stimulates autophagic degradation by preventing superfluous initiation of autophagosomes, thereby promoting an efficient autophagic flux important to survive starvation.

USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth.

Ewing sarcoma is the second most common pediatric bone and soft tissue tumor presenting with an aggressive behavior and prevalence to metastasize. The diagnostic translocation t(22;11)(q24;12) leads to expression of the chimeric oncoprotein EWS-FLI1 which is uniquely expressed in all tumor cells and maintains their survival. Constant EWS-FLI1 protein turnover is regulated by the ubiquitin proteasome system. Here, we now identified ubiquitin specific protease 19 (USP19) as a regulator of EWS-FLI1 stability using an siRNA based screening approach. Depletion of USP19 resulted in diminished EWS-FLI1 protein levels and, vice versa, upregulation of active USP19 stabilized the fusion protein. Importantly, stabilization appears to be specific for the fusion protein as it could not be observed neither for EWSR1 nor for FLI1 wild type proteins even though USP19 binds to the N-terminal EWS region to regulate deubiquitination of both EWS-FLI1 and EWSR1. Further, stable shUSP19 depletion resulted in decreased cell growth and diminished colony forming capacity in vitro, and significantly delayed tumor growth in vivo. Our findings not only provide novel insights into the importance of the N-terminal EWSR1 domain for regulation of fusion protein stability, but also indicate that inhibition of deubiquitinating enzyme(s) might constitute a novel therapeutic strategy in treatment of Ewing sarcoma.

Proteasomal Degradation of the EWS-FLI1 Fusion Protein Is Regulated by a Single Lysine Residue.

E-26 transformation-specific (ETS) proteins are transcription factors directing gene expression through their conserved DNA binding domain. They are implicated as truncated forms or interchromosomal rearrangements in a variety of tumors including Ewing sarcoma, a pediatric tumor of the bone. Tumor cells express the chimeric oncoprotein EWS-FLI1 from a specific t(22;11)(q24;12) translocation. EWS-FLI1 harbors a strong transactivation domain from EWSR1 and the DNA-binding ETS domain of FLI1 in the C-terminal part of the protein. Although Ewing cells are crucially dependent on continuous expression of EWS-FLI1, its regulation of turnover has not been characterized in detail. Here, we identify the EWS-FLI1 protein as a substrate of the ubiquitin-proteasome system with a characteristic polyubiquitination pattern. Using a global protein stability approach, we determined the half-life of EWS-FLI1 to lie between 2 and 4 h, whereas full-length EWSR1 and FLI1 were more stable. By mass spectrometry, we identified two ubiquitin acceptor lysine residues of which only mutation of Lys-380 in the ETS domain of the FLI1 part abolished EWS-FLI1 ubiquitination and stabilized the protein posttranslationally. Expression of this highly stable mutant protein in Ewing cells while simultaneously depleting the endogenous wild type protein differentially modulates two subgroups of target genes to be either EWS-FLI1 protein-dependent or turnover-dependent. The majority of target genes are in an unaltered state and cannot be further activated. Our study provides novel insights into EWS-FLI1 turnover, a critical pathway in Ewing sarcoma pathogenesis, and lays new ground to develop novel therapeutic strategies in Ewing sarcoma.

PI3K/AKT signaling modulates transcriptional expression of EWS/FLI1 through specificity protein 1.

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.

PLK1 phosphorylates PAX3-FOXO1, the inhibition of which triggers regression of alveolar Rhabdomyosarcoma.

Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503, leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as a target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1-mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free survival and overall survival. Taken together, these preclinical studies validate the PLK1-PAX3-FOXO1 axis as a rational target to treat aRMS.