Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor

Inflammatory bowel diseases (IBDs), mainly Crohn's disease and ulcerative colitis, are dynamic, chronic inflammatory conditions that are associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism for regulating IBD. Peptidylarginine deiminases (PADs) catalyze the posttranslational conversion of peptidylarginine to peptidylcitrulline in a calcium-dependent, irreversible reaction and mediate the effects of proinflammatory cytokines. Because PAD levels are elevated in mouse and human colitis, we hypothesized that a novel small-molecule inhibitor of the PADs, i.e., chloramidine (Cl-amidine), could suppress colitis in a dextran sulfate sodium mouse model. Results are consistent with this hypothesis, as demonstrated by the finding that Cl-amidine treatment, both prophylactic and after the onset of disease, reduced the clinical signs and symptoms of colitis, without any indication of toxic side effects. Interestingly, Cl-amidine drives apoptosis of inflammatory cells in vitro and in vivo, providing a mechanism by which Cl-amidine suppresses colitis. In total, these data help validate the PADs as therapeutic targets for the treatment of IBD and further suggest Cl-amidine as a candidate therapy for this disease.     

Structural and biophysical insight into cholesteryl ester-transfer protein

CETP (cholesteryl ester-transfer protein) is essential for neutral lipid transfer between HDL (high-density lipoprotein) and LDL (low-density lipoprotein) and plays a critical role in the reverse cholesterol transfer pathway. In clinical trials, CETP inhibitors increase HDL levels and reduce LDL levels, and therefore may be used as a potential treatment for atherosclerosis. In this review, we cover the analysis of CETP structure and provide insights into CETP-mediated lipid transfer based on a collection of structural and biophysical data.     

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.     

Understanding apparent DNA flexibility enhancement by HU and HMGB architectural proteins

Understanding and predicting the mechanical properties of protein/DNA complexes are challenging problems in biophysics. Certain architectural proteins bind DNA without sequence specificity and strongly distort the double helix. These proteins rapidly bind and unbind, seemingly enhancing the flexibility of DNA as measured by cyclization kinetics. The ability of architectural proteins to overcome DNA stiffness has important biological consequences, but the detailed mechanism of apparent DNA flexibility enhancement by these proteins has not been clear. Here, we apply a novel Monte Carlo approach that incorporates the precise effects of protein on DNA structure to interpret new experimental data for the bacterial histone-like HU protein and two eukaryotic high-mobility group class B (HMGB) proteins binding to ∼ 200-bp DNA molecules. These data (experimental measurement of protein-induced increase in DNA cyclization) are compared with simulated cyclization propensities to deduce the global structure and binding characteristics of the closed protein/DNA assemblies. The simulations account for all observed (chain length and concentration dependent) effects of protein on DNA behavior, including how the experimental cyclization maxima, observed at DNA lengths that are not an integral helical repeat, reflect the deformation of DNA by the architectural proteins and how random DNA binding by different proteins enhances DNA cyclization to different levels. This combination of experiment and simulation provides a powerful new approach to resolve a long-standing problem in the biophysics of protein/DNA interactions.     

The inhibitory effects of chondroitin sulfate proteoglycans on oligodendrocytes

The formation of the glial scar following a spinal cord injury presents a significant barrier to the regenerative process. It is primarily composed of chondroitin sulfate proteoglycans (CSPGs) that can inhibit axonal sprouting and regeneration. Although the inhibitory effects on neurons are well documented, little is known about their effects on oligodendrocyte progenitor cells (OPCs). In this study, we examined the effects of CSPGs on OPC process outgrowth and differentiation in vitro. The results show that specific CSPGs, in particularly those highly up-regulated following spinal cord injury, inhibit OPC process outgrowth and differentiation, and that treatment with chondroitinase ABC can completely reverse this inhibition. Additionally, treatment with the Rho kinase inhibitor Y-27632 also reverses the observed inhibition, implicating the activation of Rho kinase in the CSPG inhibition of OPC growth. Taken together, these findings demonstrate that the CSPGs found within the glial scar are not only inhibitory to neurons, but also to OPCs. Moreover, this study shows that chondroitinase ABC treatment, having shown promise in promoting axonal regeneration, may also enhance remyelination.     

Unstimulated primary CD4+ T cells from HIV-1-positive elite suppressors are fully susceptible to HIV-1 entry and productive infection

Elite controllers or suppressors (ES) are a group of HIV-1-infected individuals who maintain viral loads below the limit of detection of commercial assays for many years. The mechanisms responsible for this remarkable control are under intense study, with the hope of developing therapeutic vaccines effective against HIV-1. In this study, we addressed the question of the intrinsic susceptibility of ES CD4(+) T cells to infection. While we and others have previously shown that CD4(+) T cells from ES can be infected by HIV-1 isolates in vitro, these studies were confounded by exogenous activation and in vitro culture of CD4(+) T cells prior to infection. In order to avoid the changes in chemokine receptor expression that have been associated with such exogenous activation, we infected purified CD4(+) T cells directly after isolation from the peripheral blood of ES, viremic patients, and uninfected donors. We utilized a green fluorescent protein (GFP)-expressing proviral construct pseudotyped with CCR5-tropic or CXCR4-tropic envelope to compare viral entry using a fluorescence resonance energy transfer-based, single-round virus-cell fusion assay. The frequency of productive infection was also compared by assessing GFP expression. CD4(+) T cells from ES were as susceptible as or more susceptible than cells from viremic patients and uninfected donors to HIV-1 entry and productive infection. The results of this physiological study strongly suggest that differences in HIV-1 entry and infection of CD4(+) T cells alone cannot explain the elite control of viral replication.     

Vulture flight behavior and implications for aircraft safety

Growing vulture populations represent increasing hazards to civil and military aircraft. To assess vulture flight behavior and activity patterns at the Marine Corps Air Station in Beaufort, South Carolina, we equipped 11 black vultures (Coragyps atratus) and 11 turkey vultures (Cathartes aura) with solar-powered Global Positioning System (GPS) satellite transmitters during a 2-year study (1 Oct 2006–30 Sep 2008). Turkey vultures had larger seasonal home ranges than did black vultures, and 2 turkey vultures made round-trips to Florida. Black vultures consistently spent less time in flight (8.4%) than did turkey vultures (18.9%), and black vultures flew at higher altitudes than did turkey vultures in all seasons except summer when altitudinal distributions (above ground level) did not differ. Although we recorded maximum altitudes of 1,578 m for black vultures and 1,378 for turkey vultures, most flights were low altitude. A matrix of vulture flight altitude versus time of day revealed that >60% of vulture flight activity occurred from 4 hr to 9 hr after sunrise at altitudes below 200 m. Continuation of aggressive harassment coupled with flexible training schedules to avoid times and altitudes of high vulture activity will decrease hazards to aircraft posed by these birds. © 2011 The Wildlife Society.     

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.