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The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.

Inclusions comprised of fibrils of the microtubule- (MT-) associated protein tau are found in the brains of those with Alzheimer's disease (AD) and other neurodegenerative tauopathies. The pathology that is observed in these diseases is believed to result from the formation of toxic tau oligomers or fibrils and/or from the loss of normal tau function due to its sequestration into insoluble deposits. Hence, small molecules that prevent tau oligomerization and/or fibrillization might have therapeutic value. Indeed, examples of such compounds have been published, but nearly all have properties that render them unsuitable as drug candidates. For these reasons, we conducted quantitative high-throughput screening (qHTS) of approximately 292000 compounds to identify drug-like inhibitors of tau assembly. The fibrillization of a truncated tau fragment that contains four MT-binding domains was monitored in an assay that employed complementary thioflavin T fluorescence and fluorescence polarization methods. Previously described classes of inhibitors as well as new scaffolds were identified, including novel aminothienopyridazines (ATPZs). A number of ATPZ analogues were synthesized, and structure-activity relationships were defined. Further characterization of representative ATPZ compounds showed they do not interfere with tau-mediated MT assembly, and they are significantly more effective at preventing the fibrillization of tau than the Abeta(1-42) peptide which forms AD senile plaques. Thus, the ATPZ molecules described here represent a novel class of tau assembly inhibitors that merit further development for testing in animal models of AD-like tau pathology.

Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase. The protein is a monomer of Mr 23,241 and catalyzes a single reaction. Steady-state kinetic parameters for the enzyme have been obtained. The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme. The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and active-site-specific manner. The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.

Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.

The affinity reagent N10-(bromoacetyl)-5,8-dideazafolate has previously been shown to inactivate glycinamide ribonucleotide transformylase (EC 2.1.2.2) from Escherichia coli in an active-site-directed manner with a 1:1 stoichiometry [Inglese et al. (1990) Biochemistry 29, 1436-1443]. After a series of mild proteolytic digestions, the dideazafolate label was localized to an active-site peptide attached by an ester linkage to the highly conserved residue Asp 144. Subsequent site-specific mutagenesis of Asp 144 to Asn 144 resulted in a catalytically inactive enzyme that retained the ability to bind substrates and inhibitors. The Asn 144 mutant could be further labeled with the affinity reagent in an active-site-directed stoichiometric fashion; however, the site of modification in this case was His 119. These results imply that Asp 144 may function as a general base within the catalytic center of the transformylase and is in close proximity to His 119 in the folded protein.