Adaptive Evolution of Mus Apobec3 Includes Retroviral Insertion and Positive Selection at Two Clusters of Residues Flanking the Substrate Groove

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.     

The Early Nutritional Environment of Mice Determines the Capacity for Adipose Tissue Expansion by Modulating Genes of Caveolae Structure

While the phenomenon linking the early nutritional environment to disease susceptibility exists in many mammalian species, the underlying mechanisms are unknown. We hypothesized that nutritional programming is a variable quantitative state of gene expression, fixed by the state of energy balance in the neonate, that waxes and wanes in the adult animal in response to changes in energy balance. We tested this hypothesis with an experiment, based upon global gene expression, to identify networks of genes in which expression patterns in inguinal fat of mice have been altered by the nutritional environment during early post-natal development. The effects of over- and under-nutrition on adiposity and gene expression phenotypes were assessed at 5, 10, 21 days of age and in adult C57Bl/6J mice fed chow followed by high fat diet for 8 weeks. Under-nutrition severely suppressed plasma insulin and leptin during lactation and diet-induced obesity in adult mice, whereas over-nourished mice were phenotypically indistinguishable from those on a control diet. Food intake was not affected by under- or over-nutrition. Microarray gene expression data revealed a major class of genes encoding proteins of the caveolae and cytoskeleton, including Cav1, Cav2, Ptrf (Cavin1), Ldlr, Vldlr and Mest, that were highly associated with adipose tissue expansion in 10 day-old mice during the dynamic phase of inguinal fat development and in adult animals exposed to an obesogenic environment. In conclusion gene expression profiles, fat mass and adipocyte size in 10 day old mice predicted similar phenotypes in adult mice with variable diet-induced obesity. These results are supported by phenotypes of KO mice and suggest that when an animal enters a state of positive energy balance adipose tissue expansion is initiated by coordinate changes in mRNA levels for proteins required for modulating the structure of the caveolae to maximize the capacity of the adipocyte for lipid storage.     

Polyvalent cations as permeant probes of MIC and TRPM7 pores

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 microM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.     

Generation of novel syncytium-inducing and host range variants of ecotropic moloney murine leukemia virus in Mus spicilegus

Mus spicilegus is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study, M. spicilegus neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the M. spicilegus isolates grew to equal titers on M. dunni and SC-1 cells, although MoMLV does not efficiently infect M. dunni cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SUenv. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in M. dunni cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the M. spicilegus isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in M. dunni cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in M. spicilegus. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by M. dunni.     

A novel mechanism of cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in time-dependent inhibition by nimesulide and piroxicam.     

Uncoupling protein 2, but not uncoupling protein 1, is expressed in the female mouse reproductive tract

Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane. Whereas UCP1 is uniquely present in brown adipose tissue where it uncouples respiration from ATP synthesis and activates respiration and heat production, UCP2 is present in numerous tissues, and its exact function remains to be clarified. Two sets of data provided the rationale for this study: (i) the intriguing report that UCP1 is present in uterus of mice (Nibbelink, M., Moulin, K., Arnaud, E., Duval, C., Penicaud, L., and Casteilla, L. (2001) J. Biol. Chem. 276, 47291-47295); and (ii) an observation that Ucp2(-/-) female mice (homozygous matings) have smaller litters compared with Ucp2(+/+) animals (S. Rousset and A.-M. Cassard-Doulcier, unpublished observations). These data prompted us to examine the expression of UCP1 and UCP2 in the reproductive tract of female mice. Using wild type, Ucp1(-/-) mice, and Ucp2(-/-) mice, we were unable to detect UCP1 in uterus of mice with appropriate antibodies, and we conclude that the signal assigned to UCP1 by others was neither UCP1 nor UCP2. Using a polyclonal antibody against UCP2 and tissues from Ucp2(-/-) mice as controls, UCP2 was detected in ovary, oviduct, and uterus. Expression of Ucp2 mRNA was also observed in ovary and uterus using in situ hybridization analysis. Bone marrow transplantation experiments revealed that the UCP2 signal of the ovary was restricted to ovarian cells. UCP2 level in ovary decreased during follicular growth and increased during the pre-ovulatory period, during which aspects of an inflammatory process are known to exist. Because UCP2 down-regulates reactive oxygen species, a role in the regulation of inflammatory events linked to the preparation of ovulation is suggested.