ABSTRACT
The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.
Subject(s)
Protein Binding , Proto-Oncogene Proteins p21(ras) , Ubiquitination , Humans , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Signal Transduction , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Animals , p120 GTPase Activating Protein/metabolism , p120 GTPase Activating Protein/genetics , Mice , Cell Line, Tumor , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Lysine/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , ras Proteins/metabolism , ras Proteins/genetics , Neurofibromin 1ABSTRACT
Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.
Subject(s)
Peptides , Ubiquitin-Activating Enzymes , Ubiquitin , Humans , Animals , Mice , Ubiquitination , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , MutationABSTRACT
The Nedd4 family of E3 ubiquitin ligases, consisting of a C2-WW(n)-HECT domain architecture, includes the closely related Nedd4/Nedd4-1 and Nedd4L/Nedd4-2, which play critical roles in human physiology and pathophysiology.This review focuses on the regulation of enzymatic activity of these Nedd4 proteins, as well as on their roles in regulating stability and function of membrane and other signaling proteins, such as ion channels, ion transporters, and growth factor receptors. The diseases caused by impairment of such regulation are discussed, as well as opportunities and challenges for targeting these enzymes for therapy.
Subject(s)
Endosomal Sorting Complexes Required for Transport , Ubiquitin , Humans , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Nedd4 Ubiquitin Protein Ligases/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Establishment of final leaf size in plants relies on the precise regulation of two interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here we used a yeast two-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the two proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP-expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knock-out mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.
ABSTRACT
Mutant protein aggregation and misfolding is often correlated with toxicity in neurodegenerative diseases. Aggregate-prone proteins are tagged by ubiquitin that signals them for destruction by the proteasome or autophagy, two key pathways for protein degradation and proteostasis. Here, we review recent studies showing that the regulation of aggregate-prone proteins by ubiquitin signaling is more complex than initially postulated. We discuss how the ubiquitin code of aggregate-prone proteins is written by specific E3 ubiquitin ligases and edited by deubiquitylating enzymes (DUBs) in cells and in brain tissues, as well as how this affects protein degradation. These studies have advanced our understanding of the specificity of the ubiquitin system and provide new information about its relevance to neurodegenerative diseases and therapy.
Subject(s)
Proteins/metabolism , Ubiquitin/metabolism , Animals , Humans , Models, Molecular , Protein Aggregates , Proteins/chemistry , Proteins/genetics , Proteolysis , Signal Transduction , Ubiquitin/chemistryABSTRACT
HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.
Subject(s)
Bacteriophage HK022/genetics , Genetic Therapy , Integrases/genetics , Recombination, Genetic/genetics , Bacteriophage HK022/enzymology , DNA Nucleotidyltransferases/genetics , Genome, Human/genetics , HumansABSTRACT
Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Microfilament Proteins/metabolism , Potassium Channels, Voltage-Gated/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Multimerization , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Humans , Microfilament Proteins/chemistry , Nedd4 Ubiquitin Protein Ligases , Potassium Channels, Voltage-Gated/chemistry , Proteasome Endopeptidase Complex/chemistry , Receptor, Fibroblast Growth Factor, Type 1/chemistry , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
One of the challenges encountered by the protein quality control machinery is the need to ensure that members of multiprotein complexes are available in the correct proportions. In this study, we demonstrate that the ubiquitin proteasome system (UPS) mediates the degradation of Apc11, the catalytic core subunit of the anaphase promoting complex/cyclosome (APC/C). In vitro studies have shown that Apc11, together with its E2 enzyme, is sufficient to ubiquitinate substrates independently of the APC/C. Here, we establish that this can occur in living yeast cells. We show that the tight controls regulating the function of the fully assembled APC/C can be circumvented when its substrates are ubiquitinated by the excess levels of Apc11 independently of the assembled complex. We thus suggest that the UPS-mediated degradation of Apc11 is an overlooked mechanism ensuring that proper function of the APC/C is limited to suitably delimited holoenzymes and that an imbalance in protein expression may result in detrimental gain-of-function activity, rather than merely the disruption of protein complex stoichiometry.-Volpe, M., Levinton, N., Rosenstein, N., Prag, G., Ben-Aroya, S. Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/metabolism , Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Anaphase-Promoting Complex-Cyclosome/genetics , Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome/genetics , Cell Cycle , Gene Expression Regulation, Fungal , Humans , Protein Binding , Saccharomyces cerevisiae Proteins/genetics , Substrate Specificity , Ubiquitin/metabolism , UbiquitinationABSTRACT
Global identification of substrates for PTMs (post-translational modifications) represents a critical but yet dauntingly challenging task in understanding biology and disease pathology. Here we presented a synthetic biology approach, namely 'YESS', which coupled Y2H (yeast two hybrid) interactome screening with PTMs reactions reconstituted in bacteria for substrates identification and validation, followed by the functional validation in mammalian cells. Specifically, the sequence-independent Gateway® cloning technique was adopted to afford simultaneous transfer of multiple hit ORFs (open reading frames) between the YESS sub-systems. In proof-of-evidence applications of YESS, novel substrates were identified for UBE3A and UFL1, the E3 ligases for ubiquitination and ufmylation, respectively. Therefore, the YESS approach could serve as a potentially powerful tool to study cellular signaling mediated by different PTMs.
Subject(s)
Synthetic Biology/methods , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Cell Line , Cloning, Molecular , HEK293 Cells , HeLa Cells , Humans , Open Reading Frames , Protein Processing, Post-Translational , Signal Transduction , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/geneticsABSTRACT
About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.
Subject(s)
Deubiquitinating Enzymes/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Selection, Genetic , Thiolester Hydrolases/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/metabolism , Models, Molecular , Plasmids , Signal Transduction/genetics , Ubiquitin/metabolismABSTRACT
The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K(d) of 119 µM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 µM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.
Subject(s)
Infectious Anemia Virus, Equine/metabolism , Multienzyme Complexes , Mutation , Ubiquitin-Protein Ligases , Virus Release/physiology , Animals , Humans , Infectious Anemia Virus, Equine/genetics , Mice , Models, Biological , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Protein Structure, Tertiary , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.
Subject(s)
Escherichia coli/metabolism , Protein Processing, Post-Translational/physiology , Synthetic Biology/methods , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin/metabolism , Ubiquitination/physiology , Adaptor Proteins, Vesicular Transport/metabolism , Affinity Labels , Cloning, Molecular/methods , Fluorescence Resonance Energy Transfer , Genetic Vectors/genetics , Guanine Nucleotide Exchange Factors , Guanosine Diphosphate/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Vesicular Transport Proteins , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Specific lysine residues on the ubiquitin surface were selected during the course of evolution to form different polyubiquitin chain structures that signal diverse cellular processes. A vast number of ubiquitin receptors specifically recognize and decode the signals conferred by these polyubiquitin chains. The mechanisms of formation and the structure of Lys11-linked ubiquitin, which signals for cell-cycle and innate immune control, have been elucidated. Here, we present a new crystal structure of monomeric ubiquitin that accurately mimics one of the structures of Lys11-linked ubiquitin. Analysis of the ubiquitin:ubiquitin interface demonstrates structural fitness and specificity. The interaction is exclusively hydrophilic, leaving the Ile44 hydrophobic patch, a major recognition site for ubiquitin receptors, exposed. These noncovalent ubiquitin:ubiquitin interactions are nearly identical to those reported for Lys11-linked ubiquitin and seem to play a significant role in stabilizing the crystal structure without the isopeptide bond. In vitro cross-linking analysis with wild-type ubiquitin or its mutants partially mimics the interactions in the crystal. We suggest that these interactions may play a biological role in transmitting Lys11-linked ubiquitin chain-type cellular signals.
Subject(s)
Multiprotein Complexes/chemistry , Protein Multimerization , Ubiquitin/chemistry , Amino Acid Substitution , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutation, Missense , Signal Transduction , Structure-Activity Relationship , Ubiquitin/genetics , Ubiquitin/metabolismABSTRACT
Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFß-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of ß-TrCP. Thus, the SCFSlimb/ß-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.
Subject(s)
Centrioles , Ubiquitin , Humans , Centrioles/metabolism , Ubiquitin/metabolism , beta-Transducin Repeat-Containing Proteins/genetics , beta-Transducin Repeat-Containing Proteins/metabolism , Cell Cycle , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine.
Subject(s)
Dithiothreitol , Multiple Myeloma , Proteomics , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Humans , Dithiothreitol/chemistry , Proteomics/methods , Proteome/analysis , Proteome/metabolismABSTRACT
Sleep disturbances are common among children with neurodevelopmental disorders. Here, we report a syndrome characterized by prenatal microcephaly, intellectual disability and severe disruption of sleep-wake cycles in a consanguineous family. Exome sequencing revealed homozygous variants (c.5224G>A and c.6506G>T) leading to the missense mutations E1742K and G2169V in integrator complex subunit 1 (INTS1), the core subunit of the Integrator complex. Conservation and structural analyses suggest that G2169V has a minor impact on the structure and function of the complex, while E1742K significantly alters a negatively charged conserved patch on the surface of the protein. The severe sleep-wake cycles disruption in human carriers highlights a new aspect of Integrator complex impairment. To further study INTS1 pathogenicity, we generated Ints1-deficient zebrafish lines. Mutant zebrafish larvae displayed abnormal circadian rhythms of locomotor activity and sleep, as is the case with the affected humans. Furthermore, Ints1-deficent larvae exhibited elevated levels of dopamine ß-hydroxylase (dbh) mRNA in the locus coeruleus, a wakefulness-inducing brainstem center. Altogether, these findings suggest a significant, likely indirect, effect of INTS1 and the Integrator complex on maintaining circadian rhythms of locomotor activity and sleep homeostasis across vertebrates.
Subject(s)
Circadian Rhythm , Sleep , Zebrafish , Animals , Zebrafish/genetics , Humans , Female , Male , Circadian Rhythm/genetics , Sleep/genetics , Sleep/physiology , Wakefulness/physiology , Wakefulness/genetics , Larva/genetics , Pedigree , Amino Acid Sequence , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , ChildABSTRACT
The yeast Vps27/Hse1 complex and the homologous mammalian Hrs/STAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal-sorting pathway. The Vps27/Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27/Hse1 complex at 3.0 A resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled coil, forming a 90 A-long barbell-like structure. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27/Hse1 complex on a membrane show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.
Subject(s)
Receptors, Cytoplasmic and Nuclear/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Vesicular Transport Proteins/chemistry , Amino Acid Sequence , Chromatography, Gel , Crystallography, X-Ray , Endosomal Sorting Complexes Required for Transport , Immunoprecipitation , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation/genetics , Protein Binding , Protein Structure, Tertiary , Protein Transport , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Ubiquitin/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6â Å and a diffraction limit of 2.3â Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS-PAGE analysis of dissolved crystals.
Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Vesicular Transport Proteins/chemistry , Cloning, Molecular , Crystallization , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/isolation & purificationABSTRACT
Protein ubiquitylation controls nearly all cellular pathways in eukaryotes. A repertoire of proteins named ubiquitin (Ub) receptors harbouring ubiquitin-binding domains (UBDs) recognize ubiquitylated proteins. These Ub receptors decode the Ub signal by tethering a UBD or UBDs to a functional domain or domains, thus linking the ubiquitylated target to a specific function. The rapid dynamics of ubiquitylation/deubiquitylation has impeded the characterization of ubiquitylated proteins. To bypass this obstacle, a recently developed synthetic system that reconstructs the entire eukaryotic ubiquitylation cascade in Escherichia coli was used to purify the mono-ubiquitylated form of the regulatory proteasomal non-ATPase subunit (Ub-Rpn10) from Saccharomyces cerevisiae. Here, the first crystallization and data collection of Ub-Rpn10 is reported. Purified Ub-Rpn10 was crystallized in 12%(w/v) PEG 20,000, 0.1 M MES pH 6.5 and yielded thin rhombus-shaped crystals. X-ray analysis revealed that these crystals belonged to the monoclinic system C2, with unit-cell parameters a = 107.3, b = 49.7, c = 81.3 Å, α = γ = 90.0, ß = 130.5°. A full synchrotron data set has been collected, merged and scaled with a diffraction limit of 3.14 Å.
Subject(s)
Proteasome Endopeptidase Complex/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Crystallization , Proteasome Endopeptidase Complex/isolation & purification , Saccharomyces cerevisiae Proteins/isolation & purificationABSTRACT
Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions and modifications have been developed, each presenting distinctive benefits and caveats. Here, we present a comprehensive protocol for applying a split-Chloramphenicol Acetyl-Transferase (split-CAT) based system, to study protein-protein interactions and ubiquitylation in E. coli . Functional assembly of bait and prey proteins tethered to the split-CAT fragments result in antibiotic resistance and growth on selective media. We demonstrate assays for protein interactions, protein ubiquitylation, and the system response to small compound modulators. To facilitate data collection, we provide an updated Scanner Acquisition Manager Program for Laboratory Experiments (SAMPLE; https://github.com/PragLab/SAMPLE ) that can be employed to monitor the growth of various microorganisms, including E. coli and S. cerevisiae . The advantage posed by this system lies in its sensitivity to a wide range of chloramphenicol concentrations, which allows the detection of a large spectrum of protein-protein interactions, without the need for their purification. The tight linkage between binding or ubiquitylation and growth enables the estimation of apparent relative affinity, and represents the system's quantitative characteristics. Graphical abstract.