Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors

5-Hydroxytryptamine 2A (5-HT(2A)) serotonin receptors are important for a variety of functions including vascular smooth muscle contraction, platelet aggregation, and the modulation of perception, cognition, and emotion. In a search for 5-HT(2A) receptor-interacting proteins, we discovered that caveolin-1 (Cav-1), a scaffolding protein enriched in caveolae, complexes with 5-HT(2A) receptors in a number of cell types including C6 glioma cells, transfected HEK-293 cells, and rat brain synaptic membrane preparations. To address the functional significance of this interaction, we performed RNA interference-mediated knockdown of Cav-1 in C6 glioma cells, a cell type that endogenously expresses both 5-HT(2A) receptors and Cav-1. We discovered that the in vitro knockdown of Cav-1 in C6 glioma cells nearly abolished 5-HT(2A) receptor-mediated signal transduction as measured by calcium flux assays. RNA interference-mediated knockdown of Cav-1 also greatly attenuated endogenous Galpha(q)-coupled P2Y purinergic receptor-mediated signaling without altering the signaling of PAR-1 thrombin receptors. Cav-1 appeared to modulate 5-HT(2A) signaling by facilitating the interaction of 5-HT(2A) receptors with Galpha(q). These studies provide compelling evidence for a prominent role of Cav-1 in regulating the functional activity of not only 5-HT(2A) serotonin receptors but also selected Galpha(q)-coupled receptors.     

Absence of immunodominant anti-Gag p17 (SL9) responses among Gag CTL-positive, HIV-uninfected vaccine recipients expressing the HLA-A*0201 allele

According to a number of previous reports, control of HIV replication in humans appears to be linked to the presence of anti-HIV-1 Gag-specific CD8 responses. During the chronic phase of HIV-1 infection, up to 75% of the HIV-infected individuals who express the histocompatibility leukocyte Ag (HLA)-A*0201 recognize the Gag p17 SLYNTVATL (aa residues 77-85) epitope (SL9). However, the role of the anti-SL9 CD8 CTL in controlling HIV-1 infection remains controversial. In this study we determined whether the pattern of SL9 immunodominance in uninfected, HLA-A*0201 HIV vaccine recipients is similar to that seen in chronically HIV-infected subjects. The presence of anti-SL9 responses was determined using a panel of highly sensitive cellular immunoassays, including peptide:MHC tetramer binding, IFN-gamma ELISPOT, and cytokine flow cytometry. Thirteen HLA-A*0201 vaccinees with documented anti-Gag CD8 CTL reactivities were tested, and none had a detectable anti-SL9 response. These findings strongly suggest that the pattern of SL9 epitope immunodominance previously reported among chronically infected, HLA-A*0201-positive patients is not recapitulated in noninfected recipients of Gag-containing canarypox-based candidate vaccines and may be influenced by the relative immunogenicity of these constructs.     

Replacement of staphylococcal nuclease hydrophobic core residues with those from thermophilic homologues indicates packing is improved in some thermostable proteins

The importance of tight hydrophobic core packing in stabilizing proteins found in thermophilic organisms has been vigorously disputed. Here, portions of the cores found in three thermophilic homologues were transplanted into the core of staphylococcal nuclease, a protein of modest stability. Packing of the core was evaluated by comparing interaction energy of the three mutants to the comprehensive mutant library built up previously at these same sites in staphylococcal nuclease. It was found that the interaction energy of one thermophilic sequence is extraordinarily favorable and the interaction energies of other two transplanted thermophilic sequences are good, comparable to the interaction energies of mutant cores based on cores found in mesophilic homologues. As expected when transferring just a portion of the core sequence, the mutant proteins were destabilized overall relative to wild-type staphylococcal nuclease. The overall conclusion is that improvement of packing interactions is a mechanism to confer stability employed in some proteins from thermophiles, but not all.     

Crystal Structures of Trypanosomal Histidyl-tRNA Synthetase Illuminate Differences between Eukaryotic and Prokaryotic Homologs

Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.     

Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P.The proper regulation of bacterial motility is critical for the survival of bacteria in their natural environment. One such form of regulation is bacterial chemotaxis, which enables organisms to move toward more favorable niches and away from hazardous ones. Chemotaxis regulation in flagellated swimming bacteria has been well studied in model organisms such as Escherichia coli and Bacillus subtilis (2, 36). In general, environmental changes are detected and transduced to the cytoplasmic side of the cell by a transmembrane ternary signaling complex composed of methyl-accepting chemotaxis proteins (MCPs), CheW, and CheA. Typically, MCPs anchor the complex to the membrane through their two transmembrane (TM) domains. Chemical changes in the environment are detected by the periplasmic domain of an MCP, resulting in conformational changes in the conserved cytoplasmic signaling domain. These changes can modulate the activity of the CheA kinase via interactions with CheW in the signaling complex. The response regulator CheY, another essential component of the bacterial chemotaxis pathway, is a substrate of the CheA kinase that accepts a phosphate from autophosphorylated CheA. Phosphorylated CheY (CheY∼P) interacts with the flagellar motor complex to effect bacterial swimming behavior. Although the dephosphorylation of CheY∼P can occur spontaneously, it is accelerated by phosphatases such as CheZ in E. coli and CheC as well as FliY in B. subtilis. The dephosphorylation of CheY∼P is critical for chemotaxis since it is one of the mechanisms for the desensitization of a stimulated chemotaxis pathway. This basic architecture of chemotaxis pathways is generally conserved in all the flagellated swimming bacteria examined to date (31, 36).Myxococcus xanthus is a gliding Gram-negative bacterium that encodes eight chemosensory systems based on the genome sequence (18, 54). This bacterium, which develops fruiting bodies under nutrient deprivation (25), is motile on surfaces by adventurous (A) and social (S) gliding motility (22). While A motility enables the movement of a cell that is well separated from others, S motility is functional only when cells are in close proximity. S motility is analogous to bacterial twitching in that both are powered by retraction of the type 4 pilus (Tfp) (24, 30, 38). M. xanthus S motility additionally requires exopolysaccharides (EPS) to function (29). For S motility, EPS on one cell is thought to provide the anchor and trigger for the retraction of Tfp from a neighboring cell, thus explaining the proximity requirement. Chemotaxis regulation in M. xanthus has also been investigated extensively (27, 54). One of the surprises from these investigations was that among the eight chemosensory systems, only Frz signal transduction plays a primary role in chemotaxis regulation and mutants in other systems have no or only specific defects in chemotaxis under certain experimental conditions.The M. xanthus Dif chemosensory system, while also important for tactic responses to certain species of phosphatidylethanolamine (PE) (8), plays a primary role in the regulation of EPS production (7, 53). difA, difC, and difE mutants produce no detectable levels of EPS, whereas difD and difG mutants overproduce EPS (3, 7, 53). Mutations in difD and difG have additive effects on EPS production but failed to suppress mutations in difE (6). Additional analysis, including the use of yeast two- and three-hybrid (Y2H and Y3H, respectively) systems, led to a working model for the regulation of EPS by the Dif system (6, 52). DifA (MCP-like), DifC (CheW-like), and DifE (CheA-like) were projected to form a ternary signaling complex as do the MCPs, CheW, and CheA in bacterial chemotaxis. DifE is proposed to be an autokinase whose activity is modulated by DifA in combination with DifC (6, 52). The output of the signaling complex is the phosphorylation of an unidentified downstream component by DifE. DifD (CheY-like) and DifG (CheC-like), negative regulators of EPS production, are proposed to be ancillary modulators of the output of DifE by partially diverting phosphate from the DifE kinase and thus away from its downstream target(s) (6). That is, DifD may accept phosphate from autophosphorylated DifE (DifE∼P) and DifG may function as a phosphatase to accelerate the autodephosphorylation of phosphorylated DifD (DifD∼P). Phosphorylation and dephosphorylation events, which are obviously critical to this model, had not been examined prior to this present report.In this study, we used purified Dif proteins expressed in E. coli to examine the autophosphorylation, phosphotransfer, and dephosphorylation properties of the Dif proteins in vitro. Our results provide strong evidence for most of the proposed biochemical and physical interactions among the Dif proteins. Necessary modifications of the model based on the results here are also discussed.     

Age-related differences in the lifestyle regularity of seniors experiencing bereavement, care-giving, insomnia, and advancement into old-old age

Compared to younger adults, seniors (> or = 60 yrs) often adopt a highly regular lifestyle, perhaps as an adaptive response to age-related changes in their sleep and circadian rhythms. At baseline, diary measures of lifestyle regularity (SRM-5) were obtained from 104 seniors of three separate groups. Thirty-three subjects were challenged by spousal bereavement or the need to care for a spouse at home with dementia (Challenged); 33 were suffering from formally diagnosed (DSM-IV) insomnia (Insomnia); and 38 were healthy, well-functioning older seniors in the second half of their eighth decade of life or later (Healthy Older). The objective of this study was to determine whether lifestyle regularity increased as a function of age within each of these three senior groups. Overall, age was significantly correlated with SRM-5 (r=0.41, p<0.001), with the SRM score increasing by 0.67 units/decade. The same was true for the Challenged and Insomnia groups, which also showed a significant correlation between SRM and age (Challenged: r=0.48, p<0.01; Insomnia: r=0.36, p<0.05), though with a slightly faster rate of SRM increase in the Challenged (0.95 units/decade) than Insomnia (0.55 units/decade) group. Perhaps there was no correlation between age and SRM (r=0.07, n.s.) in the Healthy Older group due to the small age range, although this group did have a higher overall SRM score than the other two groups (p<0.01). The study thus confirmed that the previously observed increase in lifestyle regularity over the adult lifespan persists into later life. This may represent an adaptive behavioral response that might be used in future therapeutic approaches.     

Chromosomal inversion discovered in C3H/HeJ mice

Mice of the inbred mouse strain C3H/HeJ have been shown to be homozygous for a chromosomal inversion on Chromosome (Chr) 6. The inversion encompasses about 20% of the chromosome from approximately 73 Mb to approximately 116 Mb. The importance of this finding is that linkage crosses using C3H/HeJ will show no recombination in this region of Chr 6. The inversion has no apparent effect on the phenotype of C3H/HeJ mice and its presence should not affect biological studies; however, use of C3H/HeJ mice for genetic analysis of Chr 6 should be avoided or the results interpreted with the inversion in mind. The inversion has been named In(6)1J (inversion Chr 6, Jackson 1).