Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.

The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.

Thermodynamic analysis of acetylation-dependent Pb1 bromodomain-histone H3 interactions.

An acetyl-histone peptide library was used to determine the thermodynamic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains interact with histones by binding acetylated lysines. The bromodomain used in this study, BrD3, is derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin remodeling complex. Steady-state fluorescence anisotropy was used to examine the variations in specificity and affinity that drive molecular recognition. Temperature and salt concentration dependence studies demonstrate that the hydrophobic effect is the primary driving force, consistent with lysine acetylation being required for binding. An electrostatic effect was observed in only two complexes where the acetyl-lysine was adjacent to an arginine. The large change in heat capacity determined for the specific complex suggests that the dehydrated BrD3-histone interface forms a tightly bound, high-affinity complex with the target site. These explorations into the thermodynamic driving forces that confer acetylation site-dependent BrD3-histone interactions improve our understanding of how individual bromodomains work in isolation. Furthermore, this work will permit the development of hypotheses regarding how the native Pb1, and the broader class of bromodomain proteins, directs multisubunit chromatin remodeling complexes to specific acetyl-nucleosome sites in vivo.

Kinetic analysis of acetylation-dependent Pb1 bromodomain-histone interactions.

Stopped-flow fluorescence anisotropy was used to determine the kinetic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains are acetyllysine binding motifs found in many chromatin associated proteins. Individual bromodomains were derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin-remodeling complex that has six tandem bromodomains in the amino-terminal region. The average k(on) and k(off) values for the formation of high-affinity complexes are 275 M(-1) s(-1) and 0.41 x 10(-3) s(-1), respectively. The average k(on) and k(off) values for the formation of low-affinity complexes are 119 M(-1) s(-1) and 1.42 x 10(-3) s(-1), respectively. Analysis of the on- and off-rates yields acetylation site-dependent equilibrium dissociation constants averaging 1.4 and 12.9 microM for high- and low-affinity complexes, respectively. This work represents the first examination of kinetic mechanisms of acetylation-dependent bromodomain-histone interactions.

Biochemical basis for functional ingredient design from fruits.

Functional food ingredients (nutraceuticals) in fruits range from small molecular components, such as the secondary plant products, to macromolecular entities, e.g., pectin and cellulose, that provide several health benefits. In fruits, the most visible functional ingredients are the color components anthocyanins and carotenoids. In addition, several other secondary plant products, including terpenes, show health beneficial activities. A common feature of several functional ingredients is their antioxidant function. For example, reactive oxygen species (ROS) can be oxidized and stabilized by flavonoid components, and the flavonoid radical can undergo electron rearrangement stabilizing the flavonoid radical. Compounds that possess an orthodihydroxy or quinone structure can interact with cellular proteins in the Keap1/Nrf2/ARE pathway to activate the gene transcription of antioxidant enzymes. Carotenoids and flavonoids can also exert their action by modulating the signal transduction and gene expression within the cell. Recent results suggest that these activities are primarily responsible for the health benefits associated with the consumption of fruits and vegetables.

Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions.

The human polybromo-1 protein is thought to localize the Polybromo, BRG1-associated factors chromatin-remodeling complex to kinetochores during mitosis via direct interaction of its six tandem bromodomains with acetylated nucleosomes. Bromodomains are acetyl-lysine binding modules roughly 100 amino acids in length originally found in chromatin associated proteins. Previous studies verified acetyl-histone binding by each bromodomain, but site-specificity, a central tenet of the histone code hypothesis, was not examined. Here, the acetylation site-dependence of bromodomain-histone interactions was examined using steady-state fluorescence anisotropy. Results indicate that single bromodomains bind specific acetyl-lysine sites within the histone tail with sub-micromolar affinity. Identification of duplicate target sites suggests that native Pb1 interacts with both copies of histone H3 upon nucleosome assembly. Quantitative analysis of single bromodomain-histone interactions can be used to develop hypotheses regarding the histone acetylation pattern that acts as the binding target of the native polybromo-1 protein.

Expression, purification and characterization of individual bromodomains from human Polybromo-1.

Computational analysis reveals six tandem bromodomains within the amino-terminal region of the human Polybromo-1 protein, a required subunit of the Polybromo, BRG1-associated factors chromatin remodeling complex. Bromodomains are acetyl-lysine binding modules found in many chromatin binding proteins and histone acetyltransferases. Recent in vivo studies suggest that bromodomains can both discriminate the presence of an acetyl group on a lysine side chain and locate the acetyl-lysine within a histone protein. Together, this implies that multiple bromodomains may be able to function cooperatively and recognize a specific acetylation pattern to localize remodeling complexes to specific chromatin sites. Here, the cloning, expression and bioactivity of recombinant bromodomains from the human Polybromo-1 protein is described. Individual bromodomains from Polybromo-1 were cloned from human cDNA into a pET30b expression vector enabling effective one-step purification by affinity chromatography. Due to complications, including the high number of rare codons found in the coding regions and the tendency of individually expressed domains to aggregate and misfold, bacterial expression was only achieved using a cell strain containing rare eukaryotic tRNAs. Fluorescence-based bioactivity assays were performed to determine if native binding features were retained. The present cloning, expression, and purification procedure enabled the preparation of large quantity and high yields of biologically active recombinant bromodomains from human Polybromo-1 for in vitro structure and function studies. This is the first report of recombinant active form of bromodomains obtained from PB1.