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Biomedical subjects

Michael J Ellison

Publications and source records attributed to Michael J Ellison.

12 recordsLinked to original sources
PubMed2006

The Cdc34/SCF ubiquitination complex mediates Saccharomyces cerevisiae cell wall integrity.

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.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Accommodating space, time and randomness in network simulation.

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.

Animals↗
PubMed2005

Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

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.

Amino Acid Sequence↗
PubMed2005

Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth.

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.

Amino Acids↗
PubMed2005

A single Mms2 "key" residue insertion into a Ubc13 pocket determines the interface specificity of a human Lys63 ubiquitin conjugation complex.

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.

Amino Acid Sequence↗
PubMed2005

Purification and properties of the ubiquitin-conjugating enzymes Cdc34 and Ubc13.Mms2.

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.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

A life-like virtual cell membrane using discrete automata.

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.

Cell Membrane↗
PubMed2003

Energetics and specificity of interactions within Ub.Uev.Ubc13 human ubiquitin conjugation complexes.

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.

Amino Acid Sequence↗
PubMed2003

An NMR-based model of the ubiquitin-bound human ubiquitin conjugation complex Mms2.Ubc13. The structural basis for lysine 63 chain catalysis.

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.

Amino Acid Sequence↗
PubMed2003

Cdc34 self-association is facilitated by ubiquitin thiolester formation and is required for its catalytic activity.

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.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

Roles of mouse UBC13 in DNA postreplication repair and Lys63-linked ubiquitination.

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.

Amino Acid Sequence↗