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Molecular Cell Jan 2024The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse...
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
Topics: Animals; Ubiquitin; Ubiquitin-Protein Ligases; Polyubiquitin; Signal Transduction; Proteasome Endopeptidase Complex; Ubiquitination; Mammals
PubMed: 38103558
DOI: 10.1016/j.molcel.2023.11.016 -
Biomolecules Aug 2023The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of... (Review)
Review
The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of the 26S proteasome from its ~66 individual polypeptide subunits is a highly orchestrated process requiring the concerted actions of both intrinsic elements of proteasome subunits, as well as assistance by extrinsic, dedicated proteasome assembly chaperones. With the advent of near-atomic resolution cryo-electron microscopy, it has become evident that the proteasome is a highly dynamic machine, undergoing numerous conformational changes in response to ligand binding and during the proteolytic cycle. In contrast, an appreciation of the role of conformational dynamics during the biogenesis of the proteasome has only recently begun to emerge. Herein, we review our current knowledge of proteasome assembly, with a particular focus on how conformational dynamics guide particular proteasome biogenesis events. Furthermore, we highlight key emerging questions in this rapidly expanding area.
Topics: Proteasome Endopeptidase Complex; Protein Conformation; Models, Molecular; Molecular Chaperones; Humans; Cryoelectron Microscopy; Proteolysis; Ubiquitin
PubMed: 37627288
DOI: 10.3390/biom13081223 -
Cell Reports Jul 2023The 26S proteasome comprises 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in cells exist as free 20S complexes; however, our...
The 26S proteasome comprises 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in cells exist as free 20S complexes; however, our mechanistic understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation uncouples 26S holoenzymes into 20S and 19S subcomplexes. Subcomplex affinity purification and quantitative mass spectrometry reveal that Ecm29 proteasome adaptor and scaffold (ECPAS) mediates this structural remodeling. The loss of ECPAS abrogates 26S dissociation, reducing degradation of 20S proteasome substrates, including puromycylated polypeptides. In silico modeling suggests that ECPAS conformational changes commence the disassembly process. ECPAS is also essential for endoplasmic reticulum stress response and cell survival during glucose starvation. In vivo xenograft model analysis reveals elevated 20S proteasome levels in glucose-deprived tumors. Our results demonstrate that the 20S-19S disassembly is a mechanism adapting global proteolysis to physiological needs and countering proteotoxic stress.
Topics: Humans; Proteasome Endopeptidase Complex; Cytoplasm; Proteolysis; Mass Spectrometry
PubMed: 37384533
DOI: 10.1016/j.celrep.2023.112701 -
Frontiers in Immunology 2023Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical...
Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, and , whereas one patient showed additive loss-of-function mutations in . Variants in two previously not associated proteasome genes, and , were found in a patient who also carried the founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.
Topics: Humans; Proteasome Endopeptidase Complex; Syndrome; Cytoplasm; Dermatitis
PubMed: 37600812
DOI: 10.3389/fimmu.2023.1190104 -
NAR Cancer Dec 2023Apolipoprotein B messenger RNA (mRNA) editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases cause genetic instability during cancer development....
Apolipoprotein B messenger RNA (mRNA) editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases cause genetic instability during cancer development. Elevated APOBEC3A (A3A) levels result in APOBEC signature mutations; however, mechanisms regulating A3A abundance in breast cancer are unknown. Here, we show that dysregulating the ubiquitin-proteasome system with proteasome inhibitors, including Food and Drug Administration-approved anticancer drugs, increased A3A abundance in breast cancer and multiple myeloma cell lines. Unexpectedly, elevated A3A occurs via an ∼100-fold increase in A3A mRNA levels, indicating that proteasome inhibition triggers a transcriptional response as opposed to or in addition to blocking A3A degradation. This transcriptional regulation is mediated in part through FBXO22, a protein that functions in SKP1-cullin-F-box ubiquitin ligase complexes and becomes dysregulated during carcinogenesis. Proteasome inhibitors increased cellular cytidine deaminase activity, decreased cellular proliferation and increased genomic DNA damage in an A3A-dependent manner. Our findings suggest that proteasome dysfunction, either acquired during cancer development or induced therapeutically, could increase A3A-induced genetic heterogeneity and thereby influence therapeutic responses in patients.
PubMed: 38155930
DOI: 10.1093/narcan/zcad058 -
Scientific Reports Sep 2023Proteasomes are multi-subunit complexes that specialize in protein degradation. Cancer cells exhibit a heightened dependence on proteasome activity, presumably to...
Proteasomes are multi-subunit complexes that specialize in protein degradation. Cancer cells exhibit a heightened dependence on proteasome activity, presumably to support their enhanced proliferation and other cancer-related characteristics. Here, a systematic analysis of TCGA breast cancer datasets revealed that proteasome subunit transcript levels are elevated in all intrinsic subtypes (luminal, HER2-enriched, and basal-like/triple-negative) when compared to normal breast tissue. Although these observations suggest a pan-breast cancer utility for proteasome inhibitors, our further experiments with breast cancer cell lines and patient-derived xenografts (PDX) pointed to triple-negative breast cancer (TNBC) as the most sensitive subtype to proteasome inhibition. Finally, using TNBC cells, we extended our studies to in vivo xenograft experiments. Our previous work has firmly established a cytoprotective role for the transcription factor NRF1 via its ability to upregulate proteasome genes in response to proteasome inhibition. In further support of this notion, we show here that NRF1 depletion significantly reduced tumor burden in an MDA-MB-231 TNBC xenograft mouse model treated with carfilzomib. Taken together, our results point to TNBC as a particularly vulnerable breast cancer subtype to proteasome inhibition and provide a proof-of-principle for targeting NRF1 as a viable means to increase the efficacy of proteasome inhibitors in TNBC tumors.
Topics: Animals; Humans; Mice; Cytoplasm; Disease Models, Animal; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Triple Negative Breast Neoplasms; NF-E2-Related Factor 1
PubMed: 37739987
DOI: 10.1038/s41598-023-43121-x -
International Journal of Radiation... Apr 2024Radiation therapy is a primary treatment for cancer, but radioresistance remains a significant challenge in improving efficacy and reducing toxicity. Accumulating... (Review)
Review
Radiation therapy is a primary treatment for cancer, but radioresistance remains a significant challenge in improving efficacy and reducing toxicity. Accumulating evidence suggests that deubiquitinases (DUBs) play a crucial role in regulating cell sensitivity to ionizing radiation. Traditional small-molecule DUB inhibitors have demonstrated radiosensitization effects, and novel deubiquitinase-targeting chimeras (DUBTACs) provide a promising strategy for radiosensitizer development by harnessing the ubiquitin-proteasome system. This review highlights the mechanisms by which DUBs regulate radiosensitivity, including DNA damage repair, the cell cycle, cell death, and hypoxia. Progress on DUB inhibitors and DUBTACs is summarized, and their potential radiosensitization effects are discussed. Developing drugs targeting DUBs appears to be a promising alternative approach to overcoming radioresistance, warranting further research into their mechanisms.
Topics: Humans; Antineoplastic Agents; Proteasome Inhibitors; Neoplasms; Deubiquitinating Enzymes; Radiation Tolerance
PubMed: 38092257
DOI: 10.1016/j.ijrobp.2023.12.003 -
Kidney International Dec 2023Estimated glomerular filtration rate (eGFR) impacts the concentration of plasma biomarkers confounding biomarker association studies of eGFR with reverse causation. To...
A novel multi-ancestry proteome-wide Mendelian randomization study implicates extracellular proteins, tubular cells, and fibroblasts in estimated glomerular filtration rate regulation.
Estimated glomerular filtration rate (eGFR) impacts the concentration of plasma biomarkers confounding biomarker association studies of eGFR with reverse causation. To identify biomarkers causally associated with eGFR, we performed a proteome-wide Mendelian randomization study. Genetic variants nearby biomarker coding genes were tested for association with plasma concentration of 1,161 biomarkers in a multi-ancestry sample of 12,066 participants from the Prospective Urban and Rural Epidemiological (PURE) study. Using two-sample Mendelian randomization, individual variants' effects on biomarker concentration were correlated with their effects on eGFR and kidney traits from published genome-wide association studies (GWAS). Genetically altered concentrations of 22 biomarkers were associated with eGFR above a Bonferroni-corrected significance threshold. Five biomarkers were previously identified by GWAS (UMOD, FGF5, LGALS7, NINJ1, COL18A1). Nine biomarkers were within 1 Mb of the lead GWAS variant but the gene for the biomarker was unidentified as the candidate for the GWAS signal (INHBC, TNFRSF11A, TCN2, PXN1, PRTN3, PSMD9, TFPI, ITGB6, CA3). Single-cell transcriptomic data indicated the 22 biomarkers are expressed in kidney tubules, collecting duct, fibroblasts, and immune cells. Pathway analysis showed significant enrichment of identified biomarkers in the extracellular kidney parenchyma. Thus, using genetic regulators of biomarker concentration via proteome-wide Mendelian randomization, we identified 22 biomarkers that appear to causally impact eGFR in either a beneficial or adverse manner. The current study provides rationale for novel therapeutic targets for eGFR and emphasized a role for extracellular proteins produced by tubular cells and fibroblasts for impacting eGFR.
Topics: Humans; Proteome; Genome-Wide Association Study; Glomerular Filtration Rate; Mendelian Randomization Analysis; Prospective Studies; Fibroblasts; Biomarkers; Proteasome Endopeptidase Complex; Nerve Growth Factors; Cell Adhesion Molecules, Neuronal
PubMed: 37774922
DOI: 10.1016/j.kint.2023.08.025 -
Molecules (Basel, Switzerland) Oct 2023Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we...
Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we designed and synthesized a novel series of proteolysis targeting chimeras (PROTACs) using a SHP2 allosteric inhibitor as warhead, with the goal of achieving SHP2 degradation both inside the cell and in vivo. Among these molecules, compound induces efficient degradation of SHP2 (DC = 35.2 ± 1.5 nM) in a concentration- and time-dependent manner. Mechanistic investigation illustrates that the -mediated SHP2 degradation requires the recruitment of the E3 ligase and is ubiquitination- and proteasome-dependent. shows improved anti-tumor activity in a number of cancer cell lines over its parent allosteric inhibitor. Importantly, administration of leads to a nearly complete tumor regression in a xenograft mouse model, as a result of robust SHP2 depletion and suppression of phospho-ERK1/2 in the tumor. Hence, represents the first SHP2 PROTAC molecule with excellent in vivo efficacy. It is anticipated that could serve not only as a new chemical tool to interrogate SHP2 biology but also as a starting point for the development of novel therapeutics targeting SHP2.
Topics: Humans; Animals; Mice; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Neoplasms; Cell Line; Proteasome Endopeptidase Complex; Ubiquitin-Protein Ligases; Proteolysis
PubMed: 37836790
DOI: 10.3390/molecules28196947 -
IScience Oct 2023Polyubiquitinated proteins are primarily degraded by the ubiquitin-proteasome system (UPS). Proteasomes are present both in the cytoplasm and nucleus. Here, we...
Polyubiquitinated proteins are primarily degraded by the ubiquitin-proteasome system (UPS). Proteasomes are present both in the cytoplasm and nucleus. Here, we investigated mechanisms coordinating proteasome subcellular localization and activity in a multicellular organism. We identified the nuclear protein-encoding gene as a proteasome regulator in a genome-wide RNAi screen. We demonstrate that depletion of causes nuclear accumulation of endogenous polyubiquitinated proteins in intestinal cells, concomitant with slower proteasomal degradation in this subcellular compartment. Remarkably, is essential for nuclear localization of proteasomes both in oocytes and intestinal cells but affects differentially the subcellular distribution of polyubiquitinated proteins. We further reveal that importin genetically interacts with and influences nuclear localization of a polyubiquitin-binding reporter. Our study shows that the conserved AKIR-1 is an important regulator of the subcellular function of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in proteostasis maintenance.
PubMed: 37767001
DOI: 10.1016/j.isci.2023.107886