ABSTRACT
RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.
Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligases , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/isolation & purification , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Protein Binding , Mitophagy , Mitochondria/metabolism , Mitochondria/pathology , I-kappa B Kinase/metabolismABSTRACT
While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.
Subject(s)
Histones/genetics , Protein Processing, Post-Translational/genetics , Proteins/isolation & purification , Single-Cell Analysis , Histones/isolation & purification , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitination/geneticsABSTRACT
Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.
Subject(s)
Avidin/chemistry , Fanconi Anemia Complementation Group D2 Protein , Fanconi Anemia Complementation Group Proteins , Proliferating Cell Nuclear Antigen , Ubiquitin-Protein Ligases , Ubiquitin , Animals , Fanconi Anemia Complementation Group D2 Protein/chemistry , Fanconi Anemia Complementation Group D2 Protein/isolation & purification , Fanconi Anemia Complementation Group Proteins/chemistry , Fanconi Anemia Complementation Group Proteins/isolation & purification , Humans , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/isolation & purification , Sf9 Cells , Spodoptera , Ubiquitin/chemistry , Ubiquitin/isolation & purification , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitinated Proteins/chemistry , Ubiquitinated Proteins/isolation & purificationABSTRACT
INTRODUCTION: Risk factors for carbapenemase-producing Enterobacterales (CPE) acquisition/infection and associated clinical outcomes have been evaluated in the context of clonal, species-specific outbreaks. Equivalent analyses for complex, multi-species outbreaks, which are increasingly common, are lacking. METHODS: Between December 2010 and January 2017, a case-control study of Klebsiella pneumoniae carbapenemase (KPC)-producing organism (KPCO) acquisition was undertaken using electronic health records from inpatients in a US academic medical centre and long-term acute care hospital (LTACH) with ongoing multi-species KPCO transmission despite a robust CPE screening programme. Cases had a first KPCO-positive culture >48 h after admission, and included colonizations and infections (defined by clinical records). Controls had at least two negative perirectal screens and no positive cultures. Risk factors for KPCO acquisition, first infection following acquisition, and 14-day mortality following each episode of infection were identified using multi-variable logistic regression. RESULTS: In 303 cases (89 with at least one infection) and 5929 controls, risk factors for KPCO acquisition included: longer inpatient stay, transfusion, complex thoracic pathology, mechanical ventilation, dialysis, and exposure to carbapenems and ß-lactam/ß-lactamase inhibitors. Exposure to other KPCO-colonized patients was only a risk factor for acquisition in a single unit, suggesting that direct patient-to-patient transmission did not play a major role. There were 15 species of KPCO; 61 (20%) cases were colonized/infected with more than one species. Fourteen-day mortality following non-urinary KPCO infection was 20% (20/97 episodes) and was associated with failure to achieve source control. CONCLUSIONS: Healthcare exposures, antimicrobials and invasive procedures increased the risk of KPCO colonization/infection, suggesting potential targets for infection control interventions in multi-species outbreaks. Evidence for patient-to-patient transmission was limited.
Subject(s)
Cross Infection/epidemiology , Cross Infection/microbiology , Klebsiella Infections/epidemiology , Academic Medical Centers , Adult , Aged , Aged, 80 and over , Bacterial Proteins , Carbapenems/therapeutic use , Case-Control Studies , Cross Infection/drug therapy , Female , Humans , Klebsiella Infections/drug therapy , Klebsiella Infections/genetics , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Male , Middle Aged , Risk Factors , Ubiquitin-Protein Ligases/isolation & purification , Virginia/epidemiology , beta-LactamasesABSTRACT
KEY MESSAGE: OsPUB67, a U-box E3 ubiquitin ligase, may interact with two drought tolerance negative regulators (OsRZFP34 and OsDIS1) and improve drought tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. E3 ubiquitin ligases are major components of the ubiquitination cascade and contribute to the biotic and abiotic stress response in plants. In the present study, we show that a rice drought responsive gene, OsPUB67, encoding the U-box E3 ubiquitin ligase was significantly induced by drought, salt, cold, JA, and ABA, and was expressed in nuclei, cytoplasm, and membrane systems. This distribution of expression suggests a significant role for OsPUB67 in a wide range of biological processes and abiotic stress response. Over-expression of OsPUB67 improved drought stress tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. Bimolecular fluorescence complementation assays revealed that a few E2s interacted with OsPUB67 with unique functional implications in different cell components. Further evidence showed that several E3 ubiquitin ligases interacted with OsPUB67, especially OsRZFP34 and OsDIS1, which are negative regulators of drought tolerance. This interaction on the stomata implied OsPUB67 might function as a heterodimeric ubiquitination complex in response to drought stress. Comprehensive transcriptome analysis revealed OsPUB67 participated in regulating genes involved in the abiotic stress response and transcriptional regulation in an ABA-dependent manner. Our findings revealed OsPUB67 mediated a multilayered complex drought stress tolerance mechanism.
Subject(s)
Droughts , Oryza/metabolism , Plant Proteins/metabolism , Stress, Physiological/physiology , Ubiquitin-Protein Ligases/metabolism , Acclimatization/genetics , Acclimatization/physiology , Amino Acid Sequence , Gene Expression Profiling , Gene Expression Regulation, Plant , Oryza/genetics , Phenotype , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Stomata/metabolism , Plants, Genetically Modified/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Seedlings , Sequence Alignment , Sequence Analysis, Protein , Sodium Chloride/pharmacology , Stress, Physiological/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purification , UbiquitinationABSTRACT
Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.
Subject(s)
COP9 Signalosome Complex/ultrastructure , NEDD8 Protein/ultrastructure , Peptide Hydrolases/ultrastructure , Ubiquitin-Protein Ligases/ultrastructure , Adaptor Proteins, Signal Transducing/isolation & purification , Adaptor Proteins, Signal Transducing/metabolism , Animals , COP9 Signalosome Complex/isolation & purification , COP9 Signalosome Complex/metabolism , Cryoelectron Microscopy , Intracellular Signaling Peptides and Proteins/isolation & purification , Intracellular Signaling Peptides and Proteins/metabolism , Mass Spectrometry , NEDD8 Protein/isolation & purification , NEDD8 Protein/metabolism , Peptide Hydrolases/isolation & purification , Peptide Hydrolases/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Sf9 Cells , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitin-Protein Ligases/metabolismABSTRACT
Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , GABAergic Neurons/physiology , N-Acetylglucosaminyltransferases/metabolism , Ubiquitin-Protein Ligases/metabolism , Aldicarb/pharmacology , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/isolation & purification , Chromatography, Affinity , GABAergic Neurons/drug effects , Presynaptic Terminals/metabolism , Protein Binding , Proteomics , Signal Transduction , Synaptic Transmission/drug effects , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA.
Subject(s)
Bacterial Proteins/metabolism , Mycobacterium smegmatis/enzymology , Mycobacterium tuberculosis/enzymology , Serine/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Binding Sites , Cell Line , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , Humans , Macrophages/drug effects , Macrophages/metabolism , Mutation/genetics , Nitrogen/pharmacology , Structure-Activity Relationship , Sulfones/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.
Subject(s)
Legionella pneumophila/enzymology , Ubiquitin-Protein Ligases/physiology , Amino Acid Sequence , Cloning, Molecular , HEK293 Cells , Humans , Legionella pneumophila/genetics , Legionnaires' Disease/genetics , Legionnaires' Disease/microbiology , Models, Molecular , Protein Conformation, alpha-Helical , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitination/geneticsABSTRACT
Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , DNA-Binding Proteins/genetics , Homeodomain Proteins/genetics , Leukemia, B-Cell/genetics , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/isolation & purification , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Leukemic , Homeodomain Proteins/metabolism , Humans , Ikaros Transcription Factor/genetics , Ikaros Transcription Factor/isolation & purification , Leukemia, B-Cell/pathology , Myeloid Ecotropic Viral Integration Site 1 Protein , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion/metabolism , Pre-B-Cell Leukemia Transcription Factor 1 , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Binding , Protein Interaction Maps , Proto-Oncogene Proteins/metabolism , Tandem Mass Spectrometry , Transcription Factors/genetics , Transcription Factors/isolation & purification , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.
Subject(s)
Endoplasmic Reticulum/metabolism , Gene Expression Regulation, Enzymologic/physiology , Multiprotein Complexes/metabolism , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/genetics , HEK293 Cells , Humans , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/isolation & purification , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
LRSAM1 is a typical RING-finger E3 ubiquitin ligase that plays an important role in many processes. The expression and purification of LRSAM1 from Escherichiacoli had not yet been reported. Here, strategies to clone, express and purify recombinant LRSAM1 in E. coli cells were developed. LRSAM1 was expressed with high yield as inclusion bodies and successfully recovered in soluble form by subsequent denaturation and renaturation steps. Refolded LRSAM1 was directly purified through two steps of ammonium sulfate precipitation, resulting in a purity of up to 95% and a yield of about 6 mg/L bacterial culture. Purified recombinant LRSAM1 exhibited a pH-dependent E3 ligase activity. Its ligase activity was RING-finger domain-dependent, and its ubiquitination favors K6-, K27-, K29- and K48-linkages in cooperation with UbcH5-type E2 enzymes.
Subject(s)
Escherichia coli/metabolism , Gene Expression , Ubiquitin-Protein Ligases , Escherichia coli/genetics , HeLa Cells , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purification , UbiquitinationABSTRACT
Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response.
Subject(s)
DNA Damage , DNA Glycosylases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , DNA Glycosylases/genetics , Gene Expression Regulation , HeLa Cells , Humans , Protein Binding , Radiation Tolerance/genetics , Radiation, Ionizing , Recombinant Fusion Proteins , Tripartite Motif Proteins , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases/isolation & purification , UbiquitinationABSTRACT
In mammals, gene expression is largely controlled at the transcriptional level. In response to environmental or intrinsic signaling, gene expression is often fine-tuned by epigenetic modifications, including DNA methylation and histone modifications. One such histone modification is ubiquitination that predominately occurs in mono-ubiquitinated forms on histone H2A and H2B. We recently identified and characterized a novel E3 ligase called TRIM37 that ubiquitinates H2A. This study highlights the consequence of aberrant histone ubiquitination at the promoters of tumor suppressor genes in breast cancer. Regulatory mechanism by which TRIM37 and other auxiliary proteins are involved in the initiation and progression of breast cancer is of utmost importance toward generating effective therapeutics. Here, we describe a detailed step-by-step process of carrying out in vitro ubiquitination assay using purified histone proteins or reconstituted nucleosomes and affinity-purified recombinant E3 ligase like TRIM37. These experimental procedures are largely based on our studies in mammalian cells and will be a useful tool to identify substrate for E3 ubiquitin ligase as well as characterizing new E3 ligases.
Subject(s)
Epigenesis, Genetic , Histones/metabolism , Ubiquitination , Animals , COS Cells , Chlorocebus aethiops , Chromatography, Affinity , Enzyme Assays , Histones/chemistry , Histones/isolation & purification , Nucleosomes/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitin-Protein Ligases/metabolismABSTRACT
PARK2 gene's mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.
Subject(s)
Cerebellum/metabolism , Hippocampus/metabolism , Prefrontal Cortex/metabolism , Substantia Nigra/metabolism , Ubiquitin-Protein Ligases/isolation & purification , Alternative Splicing , Animals , Cerebellum/chemistry , Gene Expression , Hippocampus/chemistry , Isoelectric Focusing , Male , Organ Specificity , Prefrontal Cortex/chemistry , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Proteomics , Rats , Rats, Sprague-Dawley , Substantia Nigra/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.
Subject(s)
NF-kappa B p50 Subunit/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Cell-Free System , Humans , Intracellular Signaling Peptides and Proteins , NF-kappa B p50 Subunit/chemistry , Neoplasms/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Structure, Tertiary , Sequence Alignment , Signal Transduction , Ubiquitin-Protein Ligases/isolation & purification , UbiquitinationABSTRACT
The functional protein microarray is a powerful and versatile systems biology and proteomics tool that allows the rapid activity profiling of thousands of proteins in parallel. We have recently developed a human proteome array, the HuProt array, which includes ~80 % of all the full-length proteins of the human proteome. In one recent application of the HuProt array, we identified numerous SUMO E3 ligase-dependent SUMOylation substrates. For many SUMO E3 ligases, only a small number of substrates have been identified and the target specificities of these ligases therefore remain poorly defined. In this protocol, we outline a method we developed using the HuProt array to screen the human proteome to identify novel SUMO E3 ligase substrates recognized by specific E3 ligases.
Subject(s)
Protein Array Analysis/methods , Proteome , Proteomics/methods , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Humans , Protein Processing, Post-Translational , Small Ubiquitin-Related Modifier Proteins/isolation & purification , Substrate Specificity , Sumoylation , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
WWP2 (WW domain-containing protein 2) is an E3 ubiquitin ligase belonging to the NEDD4-like protein family involved in various cell regulations, such as carcinogenesis, transcription control and cellular transport. Compared with homologues, WWP2 is difficult to express and no practical protocols have been developed for WWP2 preparation in large scale. Recently, domain structures of homologues of WWP2 have been determined by crystallography and NMR, but none for WWP2 has been attained. In this work, through a combination of extensive screening of â¼100 constructs, expression strategies and host systems, we have found a soluble HECT domain truncation (WHP2) of WWP2 which is amendable for preparation scale expression in Escherichia coli. We have also established a relatively simple purification process to achieve highly pure WHP2 protein by employing immobilized metal-affinity chromatography followed by salting out, ion exchange chromatography and finally, size exclusion chromatography. We are able to obtain about 60mg/L of the soluble WHP2. The identity and structure of the expressed WHP2 have been analyzed by mass spectrometry and circular dichroism. The native ability of WHP2 to bind different partners has been revealed by pull-down assay.
Subject(s)
Inclusion Bodies/chemistry , Plasmids/chemistry , Recombinant Fusion Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Amino Acid Sequence , Ammonium Sulfate/chemistry , Chromatography/methods , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Molecular Sequence Data , PTEN Phosphohydrolase/chemistry , Plasmids/metabolism , Protein Binding , Protein Refolding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Solubility , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.
Subject(s)
Nerve Tissue Proteins/metabolism , Parkinson Disease/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line, Tumor , Drosophila Proteins/genetics , Drosophila Proteins/isolation & purification , Drosophila Proteins/metabolism , Drosophila melanogaster , Humans , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Parkinson Disease/genetics , Protein Binding , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purificationABSTRACT
Bacterial virulence proteins often mimic host eukaryotic proteins to modify or disturb host cellular -pathways. Increasing lines of evidence show that many bacterial effector proteins have E3 ubiquitin ligase activity. The effector protein LubX is one such bacterial E3 ubiquitin ligase. We describe here the method to purify soluble LubX protein using GST-tag and Escherichia coli overexpression systems. Using the purified protein together with recombinant ubiquitin, E1, and E2 enzymes, ubiquitin ligase activity is analyzed by the in vitro ubiquitination assay.