FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.
Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.
Background-Airborne particulate matter has been linked to excess morbidity and mortality. Recent attention has focused on the effects of particulate exposure on cardiac autonomic control. Inhaled particulates may affect the autonomic nervous system either directly, by eliciting a sympathetic stress response, or indirectly, through inflammatory cytokines produced in the lungs and released into the circulation. Methods and Results-This longitudinal study examined the association of particulates Յ2.5 m in diameter (PM 2.5 ) with heart rate variability (HRV) in an occupational cohort (Nϭ40). Continuous monitoring of exposure and HR was performed during and away from work. PM 2.5 levels were higher than ambient levels typically reported in Boston, 0.167Ϯ3.205 mg/m 3 (geometric meanϮgeometric SD). We found a 2.66% decrease (95% CI, Ϫ3.75% to Ϫ1.58%) in the 5-minute SD of normal RR intervals (SDNN) for every 1 mg/m 3 increase in the 4-hour moving PM 2.5 average and a 1.02% increase (95% CI, 0.59% to 1.46%) in HR after adjusting for potential confounding factors. The decrease in SDNN became larger as the averaging interval increased. Conclusions-Workers experienced altered cardiac autonomic control after exposure to occupational and environmental PM 2.5 . There appears to be either a long-acting (several hours) and a short-acting (several minutes) component to the mechanism of action that may be related to the production of cytokines and the sympathetic stress response, respectively, or a cumulative effect that begins shortly after exposure begins. The clinical significance of these effects in a healthy working population is unclear.
Numerous studies show an association between particulate air pollution and adverse health effects. Particulate matter is a complex mixture of elemental carbon, ammonium, sulfates, nitrates, organic components, and metals. The mechanisms of action of particulate matter less than or equal to 2.5 micro m in mean aerodynamic diameter (PM(2.5)), as well as the constituents responsible for the observed cardiopulmonary health effects, have not been identified. In this study we focused on the association between the metallic component of PM(2.5) and cardiac autonomic function based on standard heart rate variability (HRV) measures in an epidemiologic study of boilermakers. Thirty-nine male boilermakers were monitored throughout a work shift. Each subject wore an ambulatory electrocardiogram (Holter) monitor and a personal monitor to measure PM(2.5). We used mixed-effects models to regress heart rate and SDNN index (standard deviation of the normal-to-normal) on PM(2.5) and six metals (vanadium, nickel, chromium, lead, copper, and manganese). There were statistically significant mean increases in the SDNN index of 11.30 msec and 3.98 msec for every 1 micro g/m(3) increase in the lead and vanadium concentrations, respectively, after adjusting for mean heart rate, age, and smoking status. Small changes in mean heart rate were seen with all exposure metrics. The results of this study suggest an association between exposure to airborne metals and significant alterations in cardiac autonomic function. These results extend our understanding of the adverse health effects of the metals component of ambient PM(2.5).
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