Isopeptide and Ester Bond Ubiquitination Regulate Degradation of the Human Dopamine Receptor 4

The human dopamine receptor 4 (hD4R) is a seven-transmembrane helical G protein-coupled receptor (GPCR) found in neural synaptic membranes. The neurotransmitter dopamine binds to and activates the hD4R, which is involved in central nervous system pathways that modulate cognition and circadian rhythms. The hD4R is the primary dopaminergic receptor for the atypical anti-psychotic drug clozapine, which is used to treat schizophrenia and other cognitive disorders. The hD4R gene is unique among the superfamily of GPCR-encoding genes because within the human population, it contains a variable number of tandem repeat (VNTR) exon polymorphism. Because of the VNTRs, the length of the primary structure of one of the intracellular loops of the hD4R can vary dramatically among individuals. Attempts have been made to correlate different VNTR structures with different behavioral traits – for example, a specific variant of hD4R is robustly correlated with attention deficit hyperactivity disorder. Like other GPCRs, hD4R functions at the plasma membrane by binding an extracellular ligand, in this case dopamine, to regulate an intracellular signaling cascade. The density of hD4R at the plasma membrane and its distribution within the neuron/synapse dictate the cellular response to dopamine. Despite the importance of hD4R in neuronal signaling, the molecular mechanisms regulating its cellular expression and degradation are unclear. Isopeptide ubiquitination of lysine residues on the cytoplasmic surface of various GPCRs regulates receptor abundance at the membrane by promoting protein degradation. I have studied the role of the ubiquitin-proteasome system in the cellular degradation of hD4R, and show here that hD4R protein levels are regulated through both a canonical and a non-canonical ubiquitination pathway. Site-directed mutagenesis of lysine residues, as well as mutagenesis of the atypical ubiquitin acceptors serine and threonine, led to an additive increase in mutant hD4R protein abundance in a cellular expression model. Chemical inhibition of the proteasome increased levels of the wild-type hD4R, but not the lysine, serine, and threonine null mutant. Both isopeptide ubiquitination of lysine and ester bond ubiquitination of serine and threonine were detected on hD4R in a model protein expression system using immunoprecipitation techniques. A proximity ligation assay was used to quantify isopeptide and ester bond ubiquitination in this protein expression system and to detect ubiquitination of hD4R in mouse primary cortical neurons. Together, these data support the hypothesis that hD4R is proteasomally degraded after isopeptide ubiquitination of lysine residues and ester ubiquitination of serine and threonine residues. The ubiquitination and subsequent degradation of hD4R represents a mechanism for cellular control over hD4R protein levels. While the low abundance of hD4R protein produced in heterologous expression systems has previously been limiting for biochemical and structural biology techniques, the degradation-resistant hD4R mutants presented here overcomes this limitation and may facilitate future research, including the identification of dopamine receptor interacting proteins (DRIPs). hD4R joins a small number of proteins that are known to be modified by ubiquitination via ester bonds. This work also describes novel techniques to confirm and quantify ester-bond ubiquitination for a given membrane protein within a cell

CO diffusion and desorption kinetics in CO2_2 ices

Diffusion of species in icy dust grain mantles is a fundamental process that shapes the chemistry of interstellar regions; yet measurements of diffusion in interstellar ice analogs are scarce. Here we present measurements of CO diffusion into CO$_2$ ice at low temperatures (T=11--23~K) using CO$_2$ longitudinal optical (LO) phonon modes to monitor the level of mixing of initially layered ices. We model the diffusion kinetics using Fick's second law and find the temperature dependent diffusion coefficients are well fit by an Arrhenius equation giving a diffusion barrier of 300 $\pm$ 40 K. The low barrier along with the diffusion kinetics through isotopically labeled layers suggest that CO diffuses through CO$_2$ along pore surfaces rather than through bulk diffusion. In complementary experiments, we measure the desorption energy of CO from CO$_2$ ices deposited at 11-50 K by temperature-programmed desorption (TPD) and find that the desorption barrier ranges from 1240 $\pm$ 90 K to 1410 $\pm$ 70 K depending on the CO$_2$ deposition temperature and resultant ice porosity. The measured CO-CO$_2$ desorption barriers demonstrate that CO binds equally well to CO$_2$ and H$_2$O ices when both are compact. The CO-CO$_2$ diffusion-desorption barrier ratio ranges from 0.21-0.24 dependent on the binding environment during diffusion. The diffusion-desorption ratio is consistent with the above hypothesis that the observed diffusion is a surface process and adds to previous experimental evidence on diffusion in water ice that suggests surface diffusion is important to the mobility of molecules within interstellar ices

High-affinity binding of chemokine analogs that display ligand bias at the HIV-1 coreceptor CCR5

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound

Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific <i>in Vivo</i> Bioorthogonal Ligation with <i>trans</i>-Cyclooctenes

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for <i>in vivo</i> site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, <b>1</b>, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single <b>1</b> to be placed at any location in a protein. We have demonstrated that protein containing <b>1</b> can be ligated to a conformationally strained <i>trans</i>-cyclooctene <i>in vitro</i> and <i>in vivo</i> with reaction rates significantly faster than most commonly used labeling methods