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Thomas Kodadek

Publications and source records attributed to Thomas Kodadek.

At least 19 recordsLinked to original sources
PubMed2006

The role of the proteasomal ATPases and activator monoubiquitylation in regulating Gal4 binding to promoters.

Recent studies have shown that the intersection between transcription and proteins involved in the ubiquitin-proteasome pathway encompasses both proteolytic and nonproteolytic functions. Examples of the latter type include evidence that monoubiquitylation of some transcriptional activators stimulates their activity. In addition, the proteasomal ATPases are recruited to many active promoters through binding to activators and play an important, nonproteolytic role in promoter escape and elongation. In this study, we report the discovery of a new nonproteolytic activity of the proteasome (specifically the proteasomal ATPases): the active destabilization of activator-promoter complexes. This reaction depends on the presence of an activation domain and ATP. Destabilization is inhibited in vitro and in vivo if the protein is monoubiquitylated or if ubiquitin is genetically fused to the activator. The fact that monoubiquitylated activator is resistant to the "stripping" activity of the proteasomal ATPases may explain, in part, why some activators require this modification in order to function efficiently.

Adenosine Triphosphatases↗
PubMed2006

Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins.

Chemical cross-linking is an attractive approach to map peptide-protein and protein-protein complexes. Previously, we explored 3,4-dihydroxylphenylalanine (DOPA) as a protein cross-linking agent upon periodate oxidation (Burdine, L.; Gillette, T. G.; Lin, H.-J.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 11442-11443). We report here a study on the chemistry of DOPA-protein cross-linking. First, using a peptide nucleic acid templated system, we identified the alpha-amino, epsilon-amino of Lys, imidazole of His, and thiol of Cys as functional groups capable of attacking DOPA ortho-quinone. Second, we demonstrated that periodate-induced DOPA-protein cross-linking could be carried out efficiently at neutral pH in the presence of excess aliphatic 1,2-diols such as ethylene glycol, lactose, and adenosine triphosphate. This result indicated that DOPA-protein cross-linking and 1,2-diol oxidative cleavage proceed via different mechanisms and that carbohydrates will not interfere with this process when carried out in crude cell extracts or on intact cells.

Amino Acids↗
PubMed2006

Keeping transcriptional activators under control.

Transcriptional activators need to be modulated and eventually switched off after the initial event that triggers their activation. Here, we discuss how ubiquitination of activators and their proteasome-mediated turnover are crucial steps in this process.

Animals↗
PubMed2006

Isolation and characterization of coactivator-binding peptoids from a combinatorial library.

Pharmacologic agents capable of activating the expression of specific genes would be valuable tools in biological research and could potentially be useful therapeutically. Efforts to develop a general solution to this problem have focused on the discovery of cell permeable mimics of native transcription factors comprised of linked DNA-binding and activation domain surrogates. Recently, we reported the isolation of a peptoid, called KBPo2, that binds a fragment of the mammalian coactivator CREB-binding protein (CBP). When delivered to a promoter-bound DNA-binding domain, this peptoid acted as a potent activation domain mimic in human cells. In this paper, we provide full details of the screening experiments and also report further characterization of this molecule as well as the other peptoids that came out of the screen. Of the three peptoids identified as putative CBP ligands, only KBPo2 demonstrated the necessary combination of binding affinity, specificity and cell permeability necessary to function as a potent activation domain mimic in cells. KBPo2 binds to CBP in a region different than that recognized by the native activation peptide from the transcription factor CREB.

CREB-Binding Protein↗
PubMed2006

Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo.

Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.

Chromatin Immunoprecipitation↗
PubMed2006

Widespread, but non-identical, association of proteasomal 19 and 20 S proteins with yeast chromatin.

It has recently become clear that various aspects of nucleic acid metabolism and the ubiquitin-proteasome pathway intersect in several direct and important ways. To begin to assess the scope of some of these activities in the yeast Saccharomyces cerevisiae, we assessed the physical and functional association of proteasomal proteins from both the 20 S core and 19 S regulatory particles with approximately 6400 yeast genes. Genome-wide chromatin immunoprecipitation analyses revealed that proteasome substituents are associated with the majority of yeast genes. Many of these associations correlated strongly with expression levels and the presence of RNA polymerase II. Although the data support the presence of the intact 26 S proteasome on most genes, several hundred yeast genes were cross-linked to either the 20 or 19 S complex but not both, consistent with some degree of independent function for the proteasomal subcomplexes.

Biochemistry↗
PubMed2006

Genome-wide analysis of human HSF1 signaling reveals a transcriptional program linked to cellular adaptation and survival.

Although HSF1 plays an important role in the cellular response to proteotoxic stressors, little is known about the structure and function of the human HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 to comprehensively identify genes regulated directly and indirectly by HSF1. The correlation between promoter binding and gene expression was not significant for all genes bound by HSF1, suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown. Among promoters bound by HSF1 following heat shock, a gene ontology analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network following siRNA knockdown showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitinylation and others, highlighting a complex transcriptional program regulated directly and indirectly by HSF1.

Adaptation, Physiological↗
PubMed2006

The proteasomal ATPase complex is required for stress-induced transcription in yeast.

Sug1 and Sug2 are two of six ATPases in the 19S regulatory particle of the 26S proteasome. We have shown previously that these proteins play a non-proteolytic role in the transcription of the GAL genes in yeast. In this study, we probe the requirement for these factors in stress-induced transcription in yeast. It is known that proteasomal proteolysis is not required for these events. Indeed, proteasome inhibitors strongly stimulate expression of these stress response genes. However, shifting strains carrying temperature-sensitive alleles of SUG1 and SUG2 to the restrictive temperature strongly inhibited the expression of HSP26, HSP104 and GAD1 in response to heat shock or treatment with menadione bisulfate. Furthermore, chromatin immunoprecipitation analysis revealed the recruitment of Sug1, Sug2 and Cim5 (another of the ATPases), but not 20S proteasome core proteins, to the promoters of these genes. These data show that the non-proteolytic requirement for the proteasomal ATPases extends beyond the GAL genes in yeast and includes at least the heat and oxidative stress-responsive genes.

Adenosine Triphosphatases↗
PubMed2005

Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads.

Many methods have been published by which combinatorial libraries may be screened for compounds capable of manipulating the function(s) of a target protein. One of the simplest approaches is to identify compounds in a library that bind the protein of interest, since these binding events usually occur on functionally important surfaces of the protein. These protein-binding compounds could also be of utility as protein capture agents in the construction of protein-detecting microarrays or related analytical devices and as reagents for the affinity purification of proteins from complex mixtures. In this article, we provide optimized methods for screening libraries of molecules displayed on the beads on which they were synthesized. This is a particularly convenient format for library screening for laboratories with limited budgets and modest robotics capabilities.

Chalcone↗
PubMed2005

Identification of Gal4 activation domain-binding proteins in the 26S proteasome by periodate-triggered cross-linking.

A common occurrence in biology is that a regulatory peptide, protein, or small molecule regulates the activity of a large multi-protein complex through direct interactions with a protein(s) in that complex. To characterize the direct receptor of the regulatory molecule, one would ideally like to study the native system. We report here that periodate-triggered cross-linking of catechol-containing regulatory factors, followed by two-dimensional electrophoresis and Western blotting, is an effective method for the characterization of regulatory factor--protein interactions in the context of large multi-protein complexes. We demonstrate the utility of this methodology by identifying the Rpt6/Sug1 and Rpt4/Sug2 proteins as the direct targets of transcriptional activation domains in the 26S proteasome.

Amino Acid Motifs↗
PubMed2005

Simple reporter gene-based assays for hairpin poly(amide) conjugate permeability and DNA-binding activity in living cells.

Hairpin poly(amide)s (HPs) are sequence specific DNA-binding compounds that have engendered considerable interest as potential pharmacological agents to manipulate the expression of specific genes. However, recent reports have indicated that the ability of HP conjugates to pass through cell membranes is sensitive to the cell type employed and the nature of the conjugate. Furthermore, while binding of HPs to DNA sequences in vitro is relatively well understood, packing of DNA into chromatin in living cells makes predicting the efficiency with which a given poly(amide) will bind its cognate site less certain. Previous methods to evaluate HP permeability and binding in vivo, while effective, are somewhat tedious and qualitative. We report here two related reporter gene-based assays that provide a more convenient and quantitative measure of poly(amide) permeability and DNA binding activity in living cells. We anticipate that these methods will complement existing tools and facilitate the development of HP conjugates with the desired biological activity.

Base Sequence↗
PubMed2005

Protein "fingerprinting" in complex mixtures with peptoid microarrays.

We report here that microarrays comprised of several thousand peptoids (oligo-N-substituted glycines) are useful tools for the identification of proteins via a "fingerprinting" approach. By using maltose-binding protein, glutathione S-transferase, and ubiquitin, a specific and highly reproducible pattern of binding was observed when fluorescently labeled protein was hybridized to the array. A similar pattern was obtained when binding of an unlabeled protein to the array was visualized by secondary hybridization of a labeled antibody against that protein, showing that native proteins can be identified without the requirement for prior chemical labeling. This work suggests that small-molecule microarrays might be used for more complex fingerprinting assays of potential diagnostic value.

Antibodies↗
PubMed2005

A potent transactivation domain mimic with activity in living cells.

Transcriptional coactivator-binding peptoids were isolated from a large combinatorial library. One of these molecules is shown to function as a potent activation domain surrogate in mammalian cells. Up to a 900-fold increase in expression of a Gal4-responsive reporter gene is observed when a steroid conjugate of the peptoid is incubated with HeLa cells expressing a Gal4 DNA-binding domain (DBD)-glucocorticoid receptor ligand-binding domain (GRLBD) fusion protein.

Animals↗
PubMed2005

A high-throughput assay for assessing the cell permeability of combinatorial libraries.

There is great interest in the identification of synthetic molecules that are capable of manipulating protein-protein interactions in living cells. Peptides, unlike other classes of small molecules, have binding properties appropriate for this application, but most are poorly cell permeable and sensitive to proteases. Therefore, considerable effort has been expended in the development of libraries of oligomeric peptide-like molecules. However, there are no clear-cut rules to guide the design of libraries rich in cell permeable compounds. Furthermore, currently available empirical methods to assess permeability may not accurately reflect true permeability and/or are capable of only modest throughput. We describe here an assay for assessing the relative cell permeability of synthetic molecules in the context of steroid fusions that is capable of high throughput and can be used in any transfectable cell line.

Binding, Competitive↗
PubMed2005

Genomic studies of transcription factor-DNA interactions.

A central issue in the regulation of gene expression is the physical association of transcription factors with relevant promoter sequences. Recently, technological advances have allowed researchers to analyze these processes on a genomic scale. In particular, the combination of the chromatin immunoprecipitation (ChIP) technique with microarray analysis (the 'ChIP to chip' experiment) is providing a wealth of new and surprising data on transcription factor-chromatin interactions. These advances are reviewed here. We also discuss future challenges in the area.

Animals↗
PubMed2005

Techniques: Oxidative cross-linking as an emergent tool for the analysis of receptor-mediated signalling events.

The development of methods for investigating protein-protein interactions in native complexes and in living cells is an important goal in proteomics. Chemical cross-linking represents a potentially powerful approach to this goal. In this article, we review the application of recently developed oxidative cross-linking techniques to this problem; the involved reactions, mediated by high-valent metal-chelate complexes, are highly efficient in many cases, extremely rapid and do not require chemically modified proteins, although new chemical design strategies can broaden the scope of applications. Some promising applications of this chemistry to model systems is reviewed, including the fast and convenient covalent labelling of G-protein-coupled receptors (GPCRs) in intact cells, with an emphasis on the perspectives of mapping signalling events triggered by these complexes. Progress towards resolving the outstanding problems of signalling network elucidation, in addition to chemical and analytical issues that must be addressed, are discussed.

Chemistry, Pharmaceutical↗