ABSTRACT
As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.
Subject(s)
Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/metabolism , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/metabolism , Humans , Models, Biological , Models, Molecular , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/metabolism , Protein Conformation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity , Ubiquitin/chemistry , Ubiquitin/metabolismABSTRACT
The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.
ABSTRACT
The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.
Subject(s)
Deubiquitinating Enzymes/metabolism , Serine/metabolism , Ubiquitin/metabolism , Ubiquitination/physiology , A549 Cells , Bacterial Proteins/metabolism , Catalytic Domain/physiology , Cell Line , Cell Line, Tumor , Endoplasmic Reticulum/metabolism , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions/physiology , Humans , Legionella pneumophila/pathogenicity , Legionnaires' Disease/metabolism , Vacuoles/metabolismABSTRACT
Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.
Subject(s)
Ataxin-3/metabolism , Mechanistic Target of Rapamycin Complex 1/physiology , Ras Homolog Enriched in Brain Protein/metabolism , Amino Acids/metabolism , Animals , Ataxin-3/physiology , Cell Line , Deubiquitinating Enzymes/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Monomeric GTP-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Protein Binding/physiology , Ras Homolog Enriched in Brain Protein/physiology , Repressor Proteins/metabolism , Signal Transduction/physiology , UbiquitinationABSTRACT
Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.
Subject(s)
DNA Helicases/metabolism , Lysosomes/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Biosynthesis , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA, Messenger/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA Helicases/genetics , HEK293 Cells , Humans , Poly-ADP-Ribose Binding Proteins/genetics , RNA Helicases/genetics , RNA Recognition Motif Proteins/genetics , RNA, Messenger/genetics , RNA, Ribosomal, 18S , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/genetics , Ubiquitin Thiolesterase/genetics , UbiquitinationABSTRACT
Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.
Subject(s)
Fanconi Anemia , Humans , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Lysine/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/chemistry , Fanconi Anemia Complementation Group Proteins/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/chemistry , Fanconi Anemia Complementation Group D2 Protein/metabolism , Ubiquitination , DNA/metabolism , DNA Damage , DNA RepairABSTRACT
The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/enzymology , Cell Movement , Intracellular Signaling Peptides and Proteins/metabolism , Neoplastic Stem Cells/enzymology , Phosphoproteins/metabolism , Thiolester Hydrolases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Differentiation , Cell Line, Tumor , Cell Movement/drug effects , Epidermal Growth Factor/pharmacology , ErbB Receptors/agonists , ErbB Receptors/metabolism , Female , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Lysine , Mice, Inbred BALB C , Mice, Nude , Mutation , Neoplasm Metastasis , Neoplastic Stem Cells/pathology , Phenotype , Phosphoproteins/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Proteolysis , Proto-Oncogene Proteins p21(ras)/genetics , Signal Transduction , Sumoylation , Thiolester Hydrolases/genetics , Time Factors , Trans-Activators , Transcription Factors , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling ProteinsABSTRACT
In mammals, â¼100 deubiquitinases act on â¼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.
Subject(s)
BRCA1 Protein/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , Deubiquitinating Enzymes/genetics , Histone Chaperones/genetics , Neoplasms/genetics , Binding Sites/genetics , Carrier Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/immunology , Cytoplasm/genetics , Cytoplasm/immunology , DNA Breaks, Double-Stranded , DNA Repair/immunology , Deubiquitinating Enzymes/immunology , HeLa Cells , Humans , Immunity, Cellular/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Neoplasms/immunology , Nuclear Matrix-Associated Proteins/genetics , Protein Binding/genetics , Ubiquitin/genetics , Ubiquitin-Specific Proteases/genetics , Ubiquitination/geneticsABSTRACT
Transcription factor ELONGATED HYPOCOTYL5 (HY5) is the central hub for seedling photomorphogenesis. E3 ubiquitin (Ub) ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) inhibits HY5 protein accumulation through ubiquitination. However, the process of HY5 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain HY5 homeostasis has never been studied. Here, we identified that Arabidopsis thaliana deubiquitinating enzyme, Ub-SPECIFIC PROTEASE 14 (UBP14) physically interacts with HY5 and enhances its protein stability by deubiquitination. The da3-1 mutant lacking UBP14 function exhibited a long hypocotyl phenotype, and UBP14 deficiency led to the failure of rapid accumulation of HY5 during dark to light. In addition, UBP14 preferred to stabilize nonphosphorylated form of HY5 which is more readily bound to downstream target genes. HY5 promoted the expression and protein accumulation of UBP14 for positive feedback to facilitate photomorphogenesis. Our findings thus established a mechanism by which UBP14 stabilizes HY5 protein by deubiquitination to promote photomorphogenesis in A. thaliana.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Basic-Leucine Zipper Transcription Factors , Gene Expression Regulation, Plant , Ubiquitination , Arabidopsis/metabolism , Arabidopsis/growth & development , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Ubiquitin-Specific Proteases/metabolism , Ubiquitin-Specific Proteases/genetics , Protein Stability/radiation effects , Light , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Hypocotyl/growth & development , Hypocotyl/metabolism , Hypocotyl/geneticsABSTRACT
Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.
Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA-Binding Proteins , Proliferating Cell Nuclear Antigen , Ubiquitin Thiolesterase , Ubiquitin-Specific Peptidase 7 , Ubiquitination , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/genetics , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitin-Specific Peptidase 7/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Thiolester Hydrolases/metabolism , Thiolester Hydrolases/genetics , Ubiquitin/metabolism , DNA Damage , Protein Binding , Ubiquitin-Specific ProteasesABSTRACT
Hypoxia regulates tumor angiogenesis, metabolism, and therapeutic response in malignant cancers including glioblastoma, the most lethal primary brain tumor. The regulation of HIF transcriptional factors by the ubiquitin-proteasome system is critical in the hypoxia response, but hypoxia-inducible deubiquitinases that counteract the ubiquitination remain poorly defined. While the activation of ERK1/2 also plays an important role in hypoxia response, the relationship between ERK1/2 activation and HIF regulation remains elusive. Here, we identified USP33 as essential deubiquitinase that stabilizes HIF-2alpha protein in an ERK1/2-dependent manner to promote hypoxia response in cancer cells. USP33 is preferentially induced in glioma stem cells by hypoxia and interacts with HIF-2alpha, leading to its stabilization through deubiquitination. The activation of ERK1/2 upon hypoxia promoted HIF-2alpha phosphorylation, enhancing its interaction with USP33. Silencing of USP33 disrupted glioma stem cells maintenance, reduced tumor vascularization, and inhibited glioblastoma growth. Our findings highlight USP33 as an essential regulator of hypoxia response in cancer stem cells, indicating a novel potential therapeutic target for brain tumor treatment.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Brain Neoplasms , Glioma , Neoplastic Stem Cells , Ubiquitin Thiolesterase , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Brain Neoplasms/pathology , Cell Hypoxia , Glioma/pathology , Humans , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolismABSTRACT
The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a promising anticancer therapeutic target. However, resistance to BET inhibitors often occurs, and it has been linked to aberrant degradation of BRD4 protein in cancer. Here, we demonstrate that the deubiquitinase DUB3 binds to BRD4 and promotes its deubiquitination and stabilization. Expression of DUB3 is transcriptionally repressed by the NCOR2-HDAC10 complex. The NCOR2 gene is frequently deleted in castration-resistant prostate cancer patient specimens, and loss of NCOR2 induces elevation of DUB3 and BRD4 proteins in cancer cells. DUB3-proficient prostate cancer cells are resistant to the BET inhibitor JQ1 in vitro and in mice, but this effect is diminished by DUB3 inhibitory agents such as CDK4/6 inhibitor in a RB-independent manner. Our findings identify a previously unrecognized mechanism causing BRD4 upregulation and drug resistance, suggesting that DUB3 is a viable therapeutic target to overcome BET inhibitor resistance in cancer.
Subject(s)
Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 6/genetics , Endopeptidases/genetics , Gene Expression Regulation, Neoplastic , Nuclear Proteins/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Transcription Factors/genetics , Animals , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Cell Cycle Proteins , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Disease Progression , Drug Resistance, Neoplasm/genetics , Endopeptidases/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Male , Mice , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Nuclear Receptor Co-Repressor 2/deficiency , Nuclear Receptor Co-Repressor 2/genetics , Piperazines/pharmacology , Prostate/drug effects , Prostate/enzymology , Prostate/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/enzymology , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Kinase Inhibitors/pharmacology , Proteolysis , Pyridines/pharmacology , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcription, Genetic , Triazoles/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
HIF1α is one of the master regulators of the hypoxia signaling pathway and its activation is regulated by multiple post-translational modifications (PTMs). Deubiquitination mediated by deubiquitylating enzymes (DUBs) is an essential PTM that mainly modulates the stability of target proteins. USP38 belongs to the ubiquitin-specific proteases (USPs). However, whether USP38 can affect hypoxia signaling is still unknown. In this study, we used quantitative real-time PCR assays to identify USPs that can influence hypoxia-responsive gene expression. We found that overexpression of USP38 increased hypoxia-responsive gene expression, but knockout of USP38 suppressed hypoxia-responsive gene expression under hypoxia. Mechanistically, USP38 interacts with HIF1α to deubiquitinate K11-linked polyubiquitination of HIF1α at Lys769, resulting in stabilization and subsequent activation of HIF1α. In addition, we show that USP38 attenuates cellular ROS and suppresses cell apoptosis under hypoxia. Thus, we reveal a novel role for USP38 in the regulation of hypoxia signaling.
Subject(s)
Hypoxia , Signal Transduction , Humans , Cell Hypoxia/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ubiquitin-Specific Proteases/metabolism , Ubiquitination , Cell LineABSTRACT
Ubiquitin-specific proteases (USPs) are a family of multi-domain deubiquitinases (DUBs) with variable architectures, some containing regulatory auxiliary domains. Among the USP family, all occurrences of intramolecular regulation presently known are autoactivating. USP8 remains the sole exception as its putative WW-like domain, conserved only in vertebrate orthologs, is autoinhibitory. Here, we present a comprehensive structure-function analysis describing the autoinhibition of USP8 and provide evidence of the physical interaction between the WW-like and catalytic domains. The solution structure of full-length USP8 reveals an extended, monomeric conformation. Coupled with DUB assays, the WW-like domain is confirmed to be the minimal autoinhibitory unit. Strikingly, autoinhibition is only observed with the WW-like domain in cis and depends on the length of the linker tethering it to the catalytic domain. Modelling of the WW:CD complex structure and mutagenesis of interface residues suggests a novel binding site in the S1 pocket. To investigate the interplay between phosphorylation and USP8 autoinhibition, we identify AMP-activated protein kinase as a highly selective modifier of S718 in the 14-3-3 binding motif. We show that 14-3-3γ binding to phosphorylated USP8 potentiates autoinhibition in a WW-like domain-dependent manner by stabilizing an autoinhibited conformation. These findings provide mechanistic details on the autoregulation of USP8 and shed light on its evolutionary significance.
ABSTRACT
Liver cancer is notoriously refractory to conventional therapeutics. Tumor progression is governed by the interplay between tumor-promoting genes and tumor-suppressor genes. BRD4, an acetyl lysine-binding protein, is overexpressed in many cancer types, which promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains incompletely understood. In addition, understanding the regulatory mechanism of BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we investigated the potential relation between BRD4 protein level and P53, the most frequently dysregulated tumor suppressor. By analyzing the TCGA datasets, we first identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. Further investigation shows that P53 promotes BRD4 protein degradation. Mechanistically, P53 indirectly represses the transcription of USP1, a deubiquitinase, through the P21-RB1 axis. USP1 itself is also overexpressed in liver cancer and we show USP1 deubiquitinates BRD4 in vivo and in vitro, which increases BRD4 stability. With cell proliferation assays and xenograft model, we show the pro-tumor role of USP1 is partially mediated by BRD4. With functional transcriptomic analysis, we find the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB1-USP1-BRD4 axis in liver cancer.
Subject(s)
Bromodomain Containing Proteins , Cell Cycle Proteins , Liver Neoplasms , Nuclear Proteins , Transcription Factors , Ubiquitin-Specific Proteases , Humans , Bromodomain Containing Proteins/genetics , Bromodomain Containing Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Genes, Tumor Suppressor , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Retinoblastoma Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Specific Proteases/metabolismABSTRACT
Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all four core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that seven of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of two of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by the inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global genome and at local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via the reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.
Subject(s)
Genomic Instability , Histones , Homologous Recombination , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Ubiquitination , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Histones/metabolism , Histones/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Mutation , Recombinational DNA RepairABSTRACT
Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.
Subject(s)
BRCA1 Protein/genetics , Germ-Line Mutation , Loss of Function Mutation , Mutation, Missense , Neurodevelopmental Disorders/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Adolescent , BRCA1 Protein/immunology , Child , Child, Preschool , Chromatin/chemistry , Chromatin/immunology , Chromatin Assembly and Disassembly/genetics , Chromatin Assembly and Disassembly/immunology , Family , Female , Gene Expression Regulation , Heterozygote , Histones/genetics , Histones/immunology , Host Cell Factor C1/genetics , Host Cell Factor C1/immunology , Humans , Infant , Male , Neurodevelopmental Disorders/immunology , Neurodevelopmental Disorders/pathology , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/immunology , Ubiquitin/genetics , Ubiquitin/immunology , Ubiquitin Thiolesterase/deficiency , Ubiquitin Thiolesterase/immunology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/immunology , UbiquitinationABSTRACT
SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections.IMPORTANCEThe ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections.
Subject(s)
SARS-CoV-2 , Viral Proteins , Virus Diseases , Humans , Deubiquitinating Enzymes , Interferons/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Severe acute respiratory syndrome-related coronavirus/physiology , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1ß (Nsp1ß) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1ß protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (Nsp1ß) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1ß, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1ß, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.
Subject(s)
Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Female , Endopeptidases/genetics , Peptide Hydrolases/metabolism , Porcine Reproductive and Respiratory Syndrome/metabolism , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/metabolism , Swine , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus ReplicationABSTRACT
Bone marrow mesenchymal stem cells (BMSCs) possess the potential to differentiate into cartilage cells. Long noncoding RNA (lncRNAs) urothelial carcinoma associated 1 (UCA1) has been confirmed to improve the chondrogenic differentiation of marrow mesenchymal stem cells (MSCs). Herein, we further investigated the effects and underlying mechanisms of these processes. The expression of UCA1 was positively associated with chondrogenic differentiation and the knockdown of UCA1 has been shown to attenuate the expression of chondrogenic markers. RNA pull-down assay and RNA immunoprecipitation showed that UCA1 could directly bind to PARP1 protein. UCA1 could improve PARP1 protein via facilitating USP9X-mediated PARP1 deubiquitination. Then these processes stimulated the NF-κB signaling pathway. In addition, PARP1 was declined in UCA1 knockdown cells, and silencing of PARP1 could diminish the increasing effects of UCA1 on the chondrogenic differentiation from MSCs and signaling pathway activation. Collectively, these outcomes suggest that UCA1 could act as a mediator of PARP1 protein ubiquitination and develop the chondrogenic differentiation of MSCs.