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1.
Cell Signal ; 74: 109712, 2020 10.
Article in English | MEDLINE | ID: mdl-32659264

ABSTRACT

The ubiquitin (Ub)-conjugating enzyme variants (Uev) Uev1A and Mms2 interact with Ubc13 to form heterodimeric complexes with different biological functions. Uev1A-Ubc13 is involved in NF-κB activation while Mms2-Ubc13 is required for the DNA-damage response. The structural comparison of the core domains of these two Uevs reveals no obvious difference, suggesting that the amino terminal extension of Uev1A plays a critical role in the functional determination. Indeed, truncated Uev1A lacking the N-terminal extension behaves like Mms2, while a chimeric protein containing the N-terminal Uev1A fused to Mms2 functionally resembles Uev1A. Interestingly, the N-terminal extension of Uev1A also dictates whether to assemble di- or poly-Ub chains in an in vitro reaction. Both thermodynamic measurements and enzymatic assays revealed that the Uev1A N-terminal extension weakens the Uev-Ubc13 interaction; however, other means capable of causing a reduced Uev1A-Ubc13 affinity and poly-Ub chain assembly do not necessarily promote NF-κB activation, indicating that the poly-Ub chain formation is not the only component contributed by the N-terminal extension of Uev1A. The physiological relevance of the Uev1A N-terminal truncation is presented and discussed.


Subject(s)
NF-kappa B/metabolism , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Binding Sites , Cell Line, Tumor , Gene Expression Regulation , Humans , Protein Binding
2.
Genome Announc ; 1(2): e0003813, 2013 Mar 14.
Article in English | MEDLINE | ID: mdl-23516183

ABSTRACT

Staphylococcus warneri is a Gram-positive bacterium commonly found in human skin flora. The genome of a laboratory S. warneri isolate, strain SG1, was sequenced to explore its mechanism of solvent tolerance and its potential as a chassis for biofuel production.

3.
Biochem Biophys Res Commun ; 378(3): 563-8, 2009 Jan 16.
Article in English | MEDLINE | ID: mdl-19056346

ABSTRACT

Ubiquitin conjugating enzyme variants (Uev) Uev1 and Mms2 share >90% sequence identity but with distinct biological functions. Here, we report the monomeric and heterodimeric crystal structures of Uev1 and comparison with that of Mms2. Uev1 alone or in complex with Ubc13 is nearly identical with the corresponding Mms2 structures, except in one surface area containing 7/14 amino acid variations. To probe the biological significance of this unique region, we raised monoclonal antibodies specifically recognizing this region of Uev1, but not of Mms2. Epitope mapping and site-specific mutagenesis revealed at least two distinct epitopes within this region. These data collectively suggest the existence of cellular proteins capable of distinguishing Uev1 from Mms2 and directing the Ubc13-Uev complex to different pathways.


Subject(s)
Conserved Sequence , Ligases/chemistry , Transcription Factors/chemistry , Ubiquitin-Conjugating Enzymes/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Dimerization , Epitope Mapping , Humans , Ligases/genetics , Ligases/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Conformation , Sequence Deletion , Transcription Factors/genetics , Transcription Factors/immunology , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/immunology
4.
Biophys J ; 94(10): 3748-59, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18234819

ABSTRACT

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.


Subject(s)
Biopolymers/chemistry , Colloids/chemistry , Cytoplasm/chemistry , Models, Biological , Models, Chemical , Models, Molecular , Computer Simulation , Diffusion , Kinetics
5.
Hypertension ; 51(2): 412-9, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18172056

ABSTRACT

The central nervous system plays a critical role in the normal control of arterial blood pressure and in its elevation in virtually all forms of hypertension. Mitochondrial dysfunction has been increasingly associated with the development of hypertension. Therefore, we examined whether mitochondrial dysfunction occurs in the brain in hypertension and characterized it at the molecular scale. Mitochondria from whole brain and brain stem from 12-week-old spontaneously hypertensive rats with elevated blood pressure (190+/-5 mm Hg) were compared against those from age-matched normotensive (134+/-7 mm Hg) Wistar Kyoto rats (n=4 in each group). Global differential analysis using 2D electrophoresis followed by tandem mass spectrometry-based protein identification suggested a downregulation of enzymes involved in cellular energetics in hypertension. Targeted differential analysis of mitochondrial respiratory complexes using the classical blue-native SDS-PAGE/Western method and a complementary combination of sucrose-gradient ultracentrifugation/tandem mass spectrometry revealed previously unknown assembly defects in complexes I, III, IV, and V in hypertension. Interestingly, targeted examination of the brain stem, a regulator of cardiovascular homeostasis and systemic blood pressure, further showed the occurrence of mitochondrial complex I dysfunction, elevated reactive oxygen species production, decreased ATP synthesis, and impaired respiration in hypertension. Our findings suggest that in already-hypertensive spontaneously hypertensive rats, the brain respiratory complexes exhibit previously unknown assembly defects. These defects impair the function of the mitochondrial respiratory chain. This mitochondrial dysfunction localizes to the brain stem and is, therefore, likely to contribute to the development, as well as to pathophysiological complications, of hypertension.


Subject(s)
Brain/enzymology , Hypertension/complications , Hypertension/physiopathology , Mitochondrial Diseases/etiology , Multienzyme Complexes/genetics , Protein Processing, Post-Translational , Animals , Cardiovascular System/physiopathology , Doxycycline/pharmacology , Electrophoresis, Gel, Two-Dimensional , Enzyme Inhibitors/pharmacology , Homeostasis , Hypertension/enzymology , Metalloendopeptidases/antagonists & inhibitors , Mitochondrial Diseases/enzymology , Multienzyme Complexes/drug effects , Protein Processing, Post-Translational/drug effects , Proteomics , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Tandem Mass Spectrometry
6.
Mol Microbiol ; 67(3): 516-27, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18069965

ABSTRACT

The Escherichia coli CpxAR two-component signal transduction system senses and responds to extracytoplasmic stress. The cpxA101* allele was previously found to reduce F plasmid conjugation by post-transcriptional inactivation of the positive activator TraJ. Microarray analysis revealed upregulation of the protease-chaperone pair, HslVU, which was shown to degrade TraJ in an E. coli C600 cpxA101* background. Double mutants of cpxA101* and hslV or hslU restored TraJ and F conjugation to wild-type levels. The constitutive overexpression of nlpE, an outer membrane lipoprotein that induces the Cpx stress response, also led to HslVU-mediated degradation of TraJ and repression of F transfer. However, Cpx-mediated TraJ degradation appears to be growth phase-dependent, as induction of nlpE in mid-log phase cells did not appreciably alter TraJ levels. Further, His6-TraJ was sensitive to HslVU degradation in vitro only when it was purified from cells overexpressing nlpE. Thus, TraJ appears to become resistant to HslVU during normal growth, with this resistance mapping to the F transfer region. Extracytoplasmic stress prevents this modification of TraJ, leaving it susceptible to HslVU. Thus, the CpxAR stress response indirectly controls the synthesis of the F mating apparatus, a complex transenvelope type IV secretion system, by degrading TraJ.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Conjugation, Genetic , Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , F Factor , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli Proteins/genetics , Gene Deletion , Gene Expression Profiling , Lipoproteins/genetics , Lipoproteins/metabolism , Models, Biological , Oligonucleotide Array Sequence Analysis , Protein Kinases/genetics , Protein Kinases/metabolism , Regulon
7.
J Biol Chem ; 282(52): 37454-60, 2007 Dec 28.
Article in English | MEDLINE | ID: mdl-17951259

ABSTRACT

Within the ubiquitin degradation pathway, the canonical signal is a lysine 48-linked polyubiquitin chain that is assembled upon an internal lysine residue of a substrate protein. Once constructed, this ubiquitin chain becomes the principle signal for recognition and target degradation by the 26S proteasome. The mechanism by which polyubiquitin chains are assembled on a substrate protein, however, has yet to be clearly defined. In an in vitro model system, purified E2-ubiquitin thiolester was unable to catalyze the formation of polyubiquitin chains in the absence of the ubiquitin-activating enzyme E1. Mutagenesis of key residues within the E1 active site revealed that its conserved catalytic cysteine residue is essential for the formation of these chains. Moreover, inactivation of the E2 active site had no effect on the ability of E1 to catalyze ubiquitin chain formation. These findings strongly suggest E1 is responsible for not only the activation of ubiquitin but also for the direct catalytic extension of a lysine 48-linked polyubiquitin chain.


Subject(s)
Lysine/chemistry , Polyubiquitin/chemistry , Ubiquitin-Activating Enzymes/chemistry , Binding Sites , Catalysis , Escherichia coli/enzymology , Escherichia coli/metabolism , Ligases/chemistry , Mutagenesis , Protein Processing, Post-Translational , Ubiquitin/chemistry , Ubiquitin-Conjugating Enzymes/chemistry
8.
Genetics ; 174(4): 1825-39, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17028344

ABSTRACT

To identify novel functions for the Cdc34/SCF ubiquitination complex, we analyzed genomewide transcriptional profiles of cdc53-1 and cdc34-2 Saccharomyces cerevisiae mutants. This analysis revealed altered expression for several gene families, including genes involved in the regulation of cell wall organization and biosynthesis. This led us to uncover a role for the Cdc34/SCF complex in the regulation of cell wall integrity. In support of this, cdc53-1 and cdc34-2 mutants exhibit phenotypes characteristic of cell wall integrity mutants, such as SDS sensitivity and temperature-sensitive suppression by osmotic stabilizers. Examination of these mutants revealed defects in their induction of Slt2 phosphorylation, indicating defects in Pkc1-Slt2 MAPK signaling. Consistent with this, synthetic genetic interactions were observed between the genes encoding the Cdc34/SCF complex and key components of the Pck1-Slt2 MAPK pathway. Further analysis revealed that Cdc34/SCF mutants have reduced levels of active Rho1, suggesting that these defects stem from the deregulated activity of the Rho1 GTPase. Altering the activity of Rho1 via manipulation of the Rho1-GAPs LRG1 or SAC7 affected Cdc34/SCF mutant growth. Strikingly, however, deletion of LRG1 rescued the growth defects associated with Cdc34/SCF mutants, whereas deletion of SAC7 enhanced these defects. Given the differential roles that these GAPs play in the regulation of Rho1, these observations indicate the importance of coordinating Cdc34/SCF activity with specific Rho1 functions.


Subject(s)
Cell Wall/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Anaphase-Promoting Complex-Cyclosome , Cell Wall/genetics , Gene Expression Profiling , Mitogen-Activated Protein Kinases/metabolism , Oligonucleotide Array Sequence Analysis , Phosphorylation , Protein Processing, Post-Translational , SKP Cullin F-Box Protein Ligases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligases , Ubiquitins/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism
9.
Curr Opin Biotechnol ; 17(5): 493-8, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16962764

ABSTRACT

Interest in the possibility of dynamically simulating complex cellular processes has escalated markedly in recent years. This interest has been fuelled by three factors: the generally accepted value in understanding living processes as integrated systems; the dramatic increase in computational capability; and the availability of new or improved technology for making the quantitative measurements that are needed to drive and validate cellular simulations. Between the extremes of atom-scale and organism-scale simulation is a vast middle-ground requiring simulation strategies that are capable of dealing with a range of spatial, temporal and molecular abundance scales that are crucial for a comprehensive understanding of integrative cell biology. Although at an early stage, methodological improvements and the development of computational platforms provide some hope that simulations will emerge that can bridge the gap between network models and the true operation of the cell as a complex machine.


Subject(s)
Computational Biology/methods , Computer Simulation , Systems Biology/methods , Animals , Cell Physiological Phenomena , Humans , Models, Biological
10.
Methods Enzymol ; 398: 43-54, 2005.
Article in English | MEDLINE | ID: mdl-16275318

ABSTRACT

A prerequisite for structure/function studies on the ubiquitin-conjugating enzymes (Ubc) Cdc34 and Ubc13.Mms2 has been the ability to express and purify recombinant derivatives of each. This chapter describes the methods used in the expression and purification of these proteins from Escherichia coli, including variations of these protocols used to generate (35)S, (15)N, (13)C/(15)N, and seleno-L-methionine derivatives. Assays used to measure the Ub thiolester and Ub conjugation activities of these Ubcs are also described.


Subject(s)
Ligases/isolation & purification , Saccharomyces cerevisiae Proteins/isolation & purification , Ubiquitin-Conjugating Enzymes/isolation & purification , Ubiquitin-Protein Ligase Complexes/isolation & purification , Anaphase-Promoting Complex-Cyclosome , Escherichia coli/enzymology , Escherichia coli/genetics , Ligases/chemistry , Ligases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Staining and Labeling , Sulfur Radioisotopes , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/metabolism
11.
J Cell Biol ; 170(5): 745-55, 2005 Aug 29.
Article in English | MEDLINE | ID: mdl-16129784

ABSTRACT

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.


Subject(s)
Ligases/metabolism , Polyubiquitin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , DNA Damage , DNA Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , I-kappa B Kinase , Ligases/genetics , Lipopolysaccharides/metabolism , Lysine/metabolism , Macromolecular Substances , Mice , Molecular Sequence Data , NF-kappa B/metabolism , Rad51 Recombinase , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , TNF Receptor-Associated Factor 2/metabolism , TNF Receptor-Associated Factor 6/metabolism , Transcription Factors/genetics , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases
13.
In Silico Biol ; 5(2): 163-78, 2005.
Article in English | MEDLINE | ID: mdl-15972012

ABSTRACT

A framework is presented that captures the discrete and probabilistic nature of molecular transport and reaction kinetics found in a living cell as well as formally representing the spatial distribution of these phenomena. This particle or agent-based approach is computationally robust and complements established methods. Namely it provides a higher level of spatial resolution than formulations based on ordinary differential equations (ODE) while offering significant advantages in computational efficiency over molecular dynamics (MD). Using this framework, a model cell membrane has been constructed with discrete particle agents that respond to local component interactions that resemble flocking or herding behavioural cues in animals. Results from simulation experiments are presented where this model cell exhibits many of the characteristic behaviours associated with its biological counterpart such as lateral diffusion, response to osmotic pressure gradients, membrane growth and cell division. Lateral diffusion rates and estimates for the membrane modulus of elasticity derived from these simple experiments fall well within a biologically relevant range of values. More importantly, these estimates were obtained by applying a simple qualitative tuning of the model membrane. Membrane growth was simulated by injecting precursor molecules into the proto-cell at different rates and produced a variety of morphologies ranging from a single large cell to a cluster of cells. The computational scalability of this methodology has been tested and results from benchmarking experiments indicate that real-time simulation of a complete bacterial cell will be possible within 10 years.


Subject(s)
Computational Biology/methods , Models, Biological , Cell Membrane , Computer Simulation , Kinetics
14.
Mol Cell Proteomics ; 4(8): 1205-9, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15911532

ABSTRACT

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images.


Subject(s)
Amino Acids/deficiency , Bacterial Proteins/chemistry , Escherichia coli/chemistry , Escherichia coli/growth & development , Genome, Bacterial , Bacterial Proteins/isolation & purification , Databases, Protein , Electrophoresis, Gel, Two-Dimensional/methods , Open Reading Frames , Peptide Mapping/methods , Sequence Analysis/methods
15.
J Biol Chem ; 280(18): 17891-900, 2005 May 06.
Article in English | MEDLINE | ID: mdl-15749714

ABSTRACT

Human Ubc13 and Mms2 (or its homolog, Uev1) form a unique ubiquitin-conjugating enzyme (Ubc) complex that generates atypical Lys(63)-linked ubiquitin conjugates. Such conjugates are attached to specific targets that modulate the activity of various cellular processes including DNA repair, mitotic progression, and nuclear factor-kappaB signaling. Whereas Ubc13 is a typical Ubc, Mms2 is a non-catalytic Ubc variant. Substantial biochemical evidence has revealed a mechanism whereby Mms2 properly orients ubiquitin to allow for Lys(63) conjugation by Ubc13; however, how this specific Ubc13-Mms2 complex is formed and why Mms2 does not form a complex with other Ubcs have not been reported. In order to address these questions, we used a structure-based approach to design mutations and characterize the human Ubc13-Mms2 interface. We used the yeast two-hybrid assay, glutathione S-transferase pull-downs, and surface plasmon resonance to test in vivo and in vitro binding. These experiments were paired with functional complementation and ubiquitin conjugation studies to provide in vivo and in vitro functional data. The results in this study allowed us to identify important residues of the Ubc13-Mms2 interface, determine a correlation between heterodimer formation and function, and conclude why Mms2 forms a specific complex with Ubc13 but not other Ubc proteins.


Subject(s)
Ligases/genetics , Lysine/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin/genetics , Amino Acid Sequence , Humans , Ligases/chemistry , Ligases/metabolism , Lysine/chemistry , Lysine/metabolism , Molecular Sequence Data , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism
16.
Mol Cell Biol ; 23(15): 5388-400, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12861024

ABSTRACT

Using a coimmunoprecipitation strategy, we showed that the Cdc34 ubiquitin (Ub)-conjugating enzyme from Saccharomyces cerevisiae self-associates in cell lysates, thereby indicating an in vivo interaction. The ability of Cdc34 to interact with itself is not dependent on its association with the ubiquitin ligase Skp1-Cdc53/Cul1-Hrt1-F-box complex. Rather, this interaction depends upon the integrity of the Cdc34-Ub thiolester. Furthermore, several principal determinants within the Cdc34 catalytic domain, including the active-site cysteine, amino acid residues S73 and S97, and its catalytic domain insertion, also play a role in self-association. Mutational studies have shown that these determinants are functionally important in vivo and operate at the levels of both Cdc34-Ub thiolester formation and Cdc34-mediated multi-Ub chain assembly. These determinants are spatially situated in a region that is close to the active site, corresponding closely to the previously identified E2-Ub interface. These observations indicate that the formation of the Cdc34-Ub thiolester is important for Cdc34 self-association and that the interaction of Cdc34-Ub thiolesters is in turn a prerequisite for both multi-Ub chain assembly and Cdc34's essential function(s). A conclusion from these findings is that the placement of ubiquitin on the Cdc34 surface is a structurally important feature of Cdc34's function.


Subject(s)
Ligases/chemistry , Ligases/metabolism , Ubiquitin-Protein Ligase Complexes , Ubiquitin/chemistry , Anaphase-Promoting Complex-Cyclosome , Binding Sites , Catalytic Domain , Cross-Linking Reagents/pharmacology , Cysteine/chemistry , DNA Mutational Analysis , Esters/chemistry , Immunoblotting , Models, Biological , Models, Molecular , Plasmids/metabolism , Precipitin Tests , Protein Binding , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins , Temperature , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes
17.
Biochemistry ; 42(26): 7922-30, 2003 Jul 08.
Article in English | MEDLINE | ID: mdl-12834344

ABSTRACT

Lys(63)-linked polyubiquitin (poly-Ub) chains appear to play a nondegradative signaling and/or recruitment role in a variety of key eukaryotic cellular processes, including NF-kappaB signal transduction and DNA repair. A protein heterodimer composed of a catalytically active ubiquitin-conjugating enzyme (Ubc13) and its homologue (Mms2 or Uev1a) forms a catalytic scaffold upon which a noncovalently associated acceptor Ub and thiolester-linked donor Ub are oriented such that Lys(63)-linked poly-Ub chain synthesis is facilitated. In this study, we have used (1)H-(15)N nuclear magnetic resonance spectroscopy, in combination with isothermal titration calorimetry, to determine the thermodynamics and kinetics of the interactions between various components of the Lys(63)-linked poly-Ub conjugation machinery. Mms2 and Uev1a interact in vitro with acceptor Ub to form 1/1 complexes with macroscopic dissociation constants of 98 +/- 15 and 213 +/- 14 microM, respectively, and appear to bind Ub in a similar fashion. Interestingly, the Mms2.Ubc13 heterodimer associates with acceptor Ub in a 1/1 complex and binds with a dissociation constant of 28 +/- 6 microM, significantly stronger than the binding of Mms2 alone. Furthermore, a dissociation constant of 49 +/- 7 nM was determined for the interaction between Mms2 and Ubc13 using isothermal titration calorimetry. In connection with previous structural studies for this system, the thermodynamics and kinetics of acceptor Ub binding to the Mms2.Ubc13 heterodimer described in detail in this study will allow for a more thorough rationalization of the mechanism of formation of Lys(63)-linked poly-Ub chains.


Subject(s)
Ligases/chemistry , Trans-Activators/chemistry , Transcription Factors , Ubiquitin/chemistry , Amino Acid Sequence , Binding Sites , Catalysis , Dimerization , Humans , Ligases/metabolism , Lysine , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Thermodynamics , Trans-Activators/metabolism , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes
18.
J Biol Chem ; 278(15): 13151-8, 2003 Apr 11.
Article in English | MEDLINE | ID: mdl-12569095

ABSTRACT

A heterodimer composed of the catalytically active ubiquitin-conjugating enzyme hUbc13 and its catalytically inactive paralogue, hMms2, forms the catalytic core for the synthesis of an alternative type of multiubiquitin chain where ubiquitin molecules are tandemly linked to one another through a Lys-63 isopeptide bond. This type of linkage, as opposed to the more typical Lys-48-linked chains, serves as a non-proteolytic marker of protein targets involved in error-free post-replicative DNA repair and NF-kappa B signal transduction. Using a two-dimensional (1)H-(15)N NMR approach, we have mapped: 1) the interaction between the subunits of the human Ubc13.Mms2 heterodimer and 2) the interactions between each of the subunits or heterodimer with a non-covalently bound acceptor ubiquitin or a thiolester-linked donor ubiquitin. Using these NMR-derived constraints and an unbiased docking approach, we have assembled the four components of this catalytic complex into a three-dimensional model that agrees well with its catalytic function.


Subject(s)
Ligases/chemistry , Ligases/metabolism , Lysine , Ubiquitin/chemistry , Ubiquitin/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Dimerization , Humans , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Ubiquitin-Conjugating Enzymes
19.
Gene ; 285(1-2): 183-91, 2002 Feb 20.
Article in English | MEDLINE | ID: mdl-12039045

ABSTRACT

The E2 enzyme, Ubc13, and the E2 enzyme variants, Uevs, form stable, high affinity complexes for the assembly of Lys63-linked ubiquitin chains. This process is involved in error-free DNA postreplication repair, the activation of kinases in the NF-kappaB signaling pathway and possibly other cellular processes. To further investigate the roles played by Ubc13 in a whole animal model, we report here the molecular cloning of mouse UBC13 and show for the first time that a mammalian UBC13 gene is able to complement the yeast ubc13 null mutant. Furthermore, in vitro analyses and a yeast two-hybrid assay show that mUbc13 is able to form stable complexes with various Uevs. In the presence of E1 and ATP, mUbc13 forms thiolesters with ubiquitin; however, the formation of Lys63-linked di-ubiquitin and multi-ubiquitin chains is dependent on Uevs. These results suggest that the roles of UBC13 are conserved throughout eukaryotes and that the mouse is an appropriate model for the study of Ubc13-mediated Lys63-linked ubiquitin signaling pathways in humans.


Subject(s)
DNA Repair , DNA Replication/genetics , Ligases/genetics , Lysine/metabolism , Transcription Factors , Ubiquitin/metabolism , Amino Acid Sequence , Animals , DNA, Complementary/chemistry , DNA, Complementary/genetics , Evolution, Molecular , Gene Expression , Genetic Complementation Test , Humans , Ligases/metabolism , Ligases/physiology , Male , Mice , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes
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