Functional consequences of genetic variation in primates on tyrosine hydroxylase (TH) expression in vitro.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is known to contain naturally occurring genetic variation in it's promoter region that associates with a number of neuropsychological disorders. As such, examining non-coding regions is important for understanding tyrosine hydroxylase function in human health and disease. We examined approximately 2 kb upstream of the translation start site within humans and non-human primates to obtain a fine resolution map of evolutionarily and functionally relevant cis-regulatory differences. Our study investigated Macaca mulatta, Pan troglodytes, Gorilla gorilla, and Homo sapiens haplotypes using transient dual-luciferase transfection in three neuroblastoma cell lines to assay the impact of naturally occurring sequence variation on expression level. In addition to trans effects between cell lines, there are several significant expression differences between primate species, but the most striking difference was seen between human haplotypes in one cell line. Underlying this variation are numerous sequence polymorphisms, two of which influence expression within humans in a non-additive and cell line-specific manner. This study highlights functional consequences of tyrosine hydroxylase genetic variation in primates. Additionally, the results emphasize the importance of examining more than one cell line, the existence of multiple functional variants in a given promoter region and the presence of non-additive cis-interactions.

Evolution of a malaria resistance gene in wild primates.

The ecology, behaviour and genetics of our closest living relatives, the nonhuman primates, should help us to understand the evolution of our own lineage. Although a large amount of data has been amassed on primate ecology and behaviour, much less is known about the functional and evolutionary genetic aspects of primate biology, especially in wild primates. As a result, even in well-studied populations in which nongenetic factors that influence adaptively important characteristics have been identified, we have almost no understanding of the underlying genetic basis for such traits. Here, we report on the functional consequences of genetic variation at the malaria-related FY (DARC) gene in a well-studied population of yellow baboons (Papio cynocephalus) living in Amboseli National Park in Kenya. FY codes for a chemokine receptor normally expressed on the erythrocyte surface that is the known entry point for the malarial parasite Plasmodium vivax. We identified variation in the cis-regulatory region of the baboon FY gene that was associated with phenotypic variation in susceptibility to Hepatocystis, a malaria-like pathogen that is common in baboons. Genetic variation in this region also influenced gene expression in vivo in wild individuals, a result we confirmed using in vitro reporter gene assays. The patterns of genetic variation in and around this locus were also suggestive of non-neutral evolution, raising the possibility that the evolution of the FY cis-regulatory region in baboons has exhibited both mechanistic and selective parallels with the homologous region in humans. Together, our results represent the first reported association and functional characterization linking genetic variation and a complex trait in a natural population of nonhuman primates.

Thermostabilizing mutations in reovirus outer-capsid protein mu1 selected by heat inactivation of infectious subvirion particles.

The 76-kDa mu1 protein of nonfusogenic mammalian reovirus is a major component of the virion outer capsid, which contains 200 mu1 trimers arranged in an incomplete T=13 lattice. In virions, mu1 is largely covered by a second major outer-capsid protein, sigma3, which limits mu1 conformational mobility. In infectious subvirion particles, from which sigma3 has been removed, mu1 is broadly exposed on the surface and can be promoted to rearrange into a protease-sensitive and hydrophobic conformer, leading to membrane perforation or penetration. In this study, mutants that resisted loss of infectivity upon heat inactivation (heat-resistant mutants) were selected from infectious subvirion particles of reovirus strains Type 1 Lang and Type 3 Dearing. All of the mutants were found to have mutations in mu1, and the heat-resistance phenotype was mapped to mu1 by both recoating and reassortant genetics. Heat-resistant mutants were also resistant to rearrangement to the protease-sensitive conformer of mu1, suggesting that heat inactivation is associated with mu1 rearrangement, consistent with published results. Rate constants of heat inactivation were determined, and the dependence of inactivation rate on temperature was consistent with the Arrhenius relationship. The Gibbs free energy of activation was calculated with reference to transition-state theory and was found to be correlated with the degree of heat resistance in each of the analyzed mutants. The mutations are located in upper portions of the mu1 trimer, near intersubunit contacts either within or between trimers in the viral outer capsid. We propose that the mutants stabilize the outer capsid by interfering with unwinding of the mu1 trimer.

Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae).

A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N'-propylurea and N-hexadecyl-N'-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC(50) around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N'-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N'-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N'-substituents. JHEH hydrolyzes t-DPPO with a K(m) of 65.6 microM and a V(max) of 59 nmol min(-1) mg(-1) and has a substantially lower K(m) of 3.6 microM and higher V(max) of 322 nmol min(-1) mg(-1) for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation.

Thermostability of reovirus disassembly intermediates (ISVPs) correlates with genetic, biochemical, and thermodynamic properties of major surface protein mu1.

Kinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein mu1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound mu1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of mu1 as a determinant of thermostability. Intact virions, which contain final sigma3 bound to mu1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for final sigma3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.