Solution structure of a small protein containing a fluorinated side chain in the core

We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe --> F(5)-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F(5)-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl-aryl or perfluoroaryl-perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 --> F(5)-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by approximately 1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe --> F(5)-Phe mutations offer the possibility of greater tertiary structural stability from side chain-side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability.     

Targeting protein-protein interactions: lessons from p53/MDM2

The tremendous challenge of inhibiting therapeutically important protein-protein interactions has created the opportunity to extend traditional medicinal chemistry to a new class of targets and to explore nontraditional strategies. Here we review a widely studied system, the interaction between tumor suppressor p53 and its natural antagonist MDM2, for which both traditional and nontraditional approaches have been reported. This system has been a testing ground for novel proteomimetic scaffold-based strategies, i.e., for attempts to mimic the recognition surface displayed by a folded protein with unnatural oligomers. Retroinverso peptides, peptoids, terphenyls, beta-hairpins, p-oligobenzamides, beta-peptides, and miniproteins have all been explored as inhibitors of the p53/MDM2 interaction, and we focus on these oligomer-based efforts. Traditional approaches have been successful as well, and we briefly review small molecule inhibitors along with other strategies for reactivation of the p53 pathway, for comparison with oligomer- based approaches. We close with comments on an emerging dichotomy among protein-protein interaction targets.     

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.     

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary

Background  

The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR).     

A Fluorescence Polarization Assay for the Identification of Inhibitors of the p53–DM2 Protein–Protein Interaction

Improper function of the tumor suppressor protein p53 is a contributing factor in many human cancers. In normal cells, p53 acts to arrest the cell cycle in response to DNA damage or nucleotide depletion. One mechanism of regulating the amount of p53 in the cell is through the action of the Double Minute 2 protein, DM2 (also known as MDM2), which ubiquitinates p53 and targets it for proteosomal degradation. In a number of human cancers, the DM2 gene is amplified or overexpressed, leading to inadequate levels of p53 for cell cycle arrest or apoptosis. With the goal of restoring p53 function in cancers that overexpress DM2, we are developing inhibitors of the p53-DM2 protein-protein interaction that structurally mimic the N-terminal segment of p53 that binds to DM2. To assist this effort, we have devised a fluorescence polarization assay that quantifies the interaction between the N-terminal regions of both proteins in 384-well microtiter plates. Using this assay, we have demonstrated that a peptide with a nonhydrolyzable beta-amino acid substitution binds DM2 with an affinity comparable to a p53 peptide that is composed of only alpha-amino acids.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Distribution of flourescently labeled α-actinin in living and fixed fibroblasts

The distribution of flourescently labeled α-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-α-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of α-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected α-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected α-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected α-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of α-actinin appears to be incorporated into the functional pools of α-actinin within the living cell and to be utilized by the cell with fidelity.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Occupational Physical Activity and Body Mass Index: Results from the Hispanic Community Health Study / Study of Latinos

Purpose

To examine the associations between overweight/obesity and occupation among Hispanics/Latinos, the largest minority population in the U.S.

Methods

This study included 7,409 employed individuals in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), a prospective study of Hispanic/Latino individuals aged 18–74 in four communities in the U.S. We independently examined the relationships between BMI, Occupational Activity (OA), and Total Hours Worked, quantified via self-reported hours worked per week and occupation-assigned Metabolic Equivalents (METs).

Results

More than three quarters of the participants were either overweight (39.3%) or obese (37.8%). Individuals with a primary occupation and those employed in a secondary occupation worked an average of 36.8 and 14.6 hrs/wk, respectively. The overall adjusted odds for being obese compared to normal weight were 3.2% (AOR = 1.03, 95% CI 1.01, 1.05) and 14.4% (AOR = 1.14 95% Cl 1.07, 1.23) greater for each 10 MET•hrs/wk unit of increased OA, and each 10-hrs/wk unit of Total Hours Worked, respectively.

Conclusion

This study presents the first findings on the association between OA with overweight/obesity among Hispanic/Latino individuals in the U.S. Increasing OA and Total Hours Worked per week were independently associated with increasing odds of overweight/obesity suggesting that the workplace is only one part of the overall energy expenditure dynamic. Our findings point to the need to emphasize engaging employed individuals in greater levels of PA outside of the work environment to impact overweight/obesity.