Entropy-enthalpy compensation in ionic interactions probed in a zinc finger peptide

Zinc(II) and cobalt(II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a beta-sheet were examined. Previous studies revealed a narrow range of interaction free energies (<0.5 kcal/mol) between these residues. Here, isothermal titration calorimetry studies were performed, revealing a range of over 3 kcal/mol in relative binding enthalpies. Double mutant cycle analysis revealed a range of interaction enthalpies ranging from -3.1 to -3.4 kcal/mol for the Arg-Asp pair to -0.8 kcal/mol for the Lys-Glu pair. The large range of interaction enthalpies coupled with the small range of interaction free energies reveals substantial entropy-enthalpy compensation. The magnitudes of the effects are consistent with the formation of a structurally rigid Arg-Asp contact ion pair but less direct and more mobile interactions involving the other combinations.     

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P(3), the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P(3) asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.     

N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies

We examined how asparagine-linked glycans within and adjacent to the V3 loop (C2 and C3 regions) and within the immunologically silent face (V4, C4, and V5 regions) of the human immunodeficiency virus (HIV) SF612 envelope affect the viral phenotype. Five of seven potential glycosylation sites are utilized when the virus is grown in human peripheral blood mononuclear cells, with the nonutilized sites lying within the V4 loop. Elimination of glycans within and adjacent to the V3 loop renders SF162 more susceptible to neutralization by polyclonal HIV(+)-positive and simian/human immunodeficiency virus-positive sera and by monoclonal antibodies (MAbs) recognizing the V3 loop, the CD4- and CCR5-binding sites, and the extracellular region of gp41. Importantly, our studies also indicate that glycans located within the immunologically silent face of gp120, specifically the C4 and V5 regions, also conferred on SF162 resistance to neutralization by anti-V3 loop, anti-CD4 binding site, and anti-gp41 MAbs but not by antibodies targeting the coreceptor binding site. We also observed that the amino acid composition of the V4 region contributes to the neutralization phenotype of SF162 by anti-V3 loop and anti-CD4 binding site MAbs. Collectively, our data support the proposal that the glycosylation and structure of the immunologically silent face of the HIV envelope plays an important role in defining the neutralization phenotype of HIV type 1.     

Gonadal hormones masculinize and defeminize reproductive behaviors during puberty in the male Syrian hamster

Three experiments were conducted to test whether testicular hormones secreted during puberty masculinize and defeminize the expression of adult reproductive behavior. Experiment 1 tested the hypothesis that gonadal hormones during puberty masculinize behavioral responses to testosterone (T) in adulthood. Male hamsters were castrated either before puberty (noTduringP) or after puberty (TduringP). All males were implanted with a 2.5-mg T pellet 6 weeks following castration and tested once for masculine reproductive behavior 7 days after the onset of T replacement. TduringP males displayed significantly more mounts, intromissions, and ejaculations than noTduringP males. Experiment 2 tested the hypothesis that gonadal hormones during puberty defeminize behavioral responses to estrogen (EB) and progesterone (P). Eight weeks following castration, noTduringP and TduringP males were primed with EB and P and tested for lordosis behavior with a stud male. Behavioral responses of males were compared to that of ovariectomized (OVX) and hormone primed females. NoTduringP males and OVX females displayed significantly shorter lordosis latencies than TduringP males. Experiment 3 investigated whether prolonged T treatment or sexual experience could reverse the deficits in masculine behavior caused by the absence of T during puberty. Extending the T treatment from 7 to 17 days did not ameliorate the deficits in masculine behavior caused by absence of T during puberty. Similarly, when the level of sexual experience was increased from one to three tests, the deficits in masculine behavior persisted. These studies demonstrate that gonadal hormones during puberty further masculinize and defeminize neural circuits and behavioral responsiveness to steroid hormones in adulthood.     

Oxantel is an N-type (methyridine and nicotine) agonist not an L-type (levamisole and pyrantel) agonist: classification of cholinergic anthelmintics in Ascaris

Three pharmacological subtypes of cholinergic receptors have been distinguished in Ascaris suum using a muscle contraction assay and classical pharmacological techniques. The receptor subtypes are: a B-subtype (sensitive to bephenium); an L-subtype (sensitive to levamisole and pyrantel); and an N-subtype (sensitive to nicotine and methyridine). Oxantel is a cholinergic anthelmintic that was first introduced for the treatment of whipworm, Trichuris, infections in children. Here, we compare the subtype selectivity of oxantel with thenium and other cholinergic anthelmintics. We used the A. suum assay to derive pA(2) values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. pA(2) values, rather than pK(B) values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The pA(2) of oxantel was 6.58+/-0.25 for paraherquamide; 5.39+/-0.28 for 2-desoxyparaherquamide; 7.01+/-0.19 for methyllycaconitine. Comparison of pA(2) values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had pA(2)s of 7.84+/-0.41 for paraherquamide; 5.52+/-0.50 for 2-desoxyparaherquamide; 6.33+/-0.19 for methyllycaconitine. Cluster analysis placed thenium between the L-subtype agonists and the B-subtype agonist. The therapeutic significance of classification of cholinergic anthelmintics is discussed. Combination of oxantel and pyrantel would have therapeutic advantages, covering N- and L-subtypes, and so increasing spectrum of action and reducing the potential for development of resistance. Our results predict that oxantel may remain effective in some nematode isolates that have become levamisole- and pyrantel-resistant.     

Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.     

Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling

In contrast to typical membrane proteins that span the lipid bilayer via transmembrane alpha-helices, bacterial outer membrane proteins adopt a beta-barrel architecture composed of antiparallel transmembrane beta-strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for alpha-helical membrane proteins do not work for beta-barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenic Escherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane beta-strands. Based on experimental evidence, we propose that the usher consists of an N-terminal beta-barrel domain comprised of 26 beta-strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal beta-barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.     

Efficiency of mammalian selenocysteine incorporation

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into approximately 25 mammalian proteins. Two of these are cis sequences, a SECIS element in the 3'-untranslated region and a Sec codon (UGA) in the coding region. The three known trans-acting factors are a Sec-specific translation elongation factor (eEFSec), the Sec-tRNA(Sec), and a SECIS-binding protein, SBP2. Here we describe a system in which the efficiency of Sec incorporation was determined quantitatively both in vitro and in transfected cells, and in which the contribution of each of the known factors is examined. The efficiency of Sec incorporation into a luciferase reporter system in vitro is maximally 5-8%, which is 6-10 times higher than that in transfected rat hepatoma cells, McArdle 7777. In contrast, the efficiency of Sec incorporation into selenoprotein P in vitro is approximately 40%, suggesting that as yet unidentified cis-elements may regulate differential selenoprotein expression. In addition, we have found that SBP2 is the only limiting factor in rabbit reticulocyte lysate but not in transfected rat hepatoma cells where SBP2 is found to be mostly if not entirely cytoplasmic despite having a strong putative nuclear localization signal. The significance of these findings with regard to the function of known Sec incorporation factors is discussed.