Protein <Superscript>19</Superscript>F-labeling using transglutaminase for the NMR study of intermolecular interactions

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic ¹⁵N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. ¹⁹F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, ¹⁹F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic ¹⁹F-labeling readily provided NMR detection of protein-drug and protein–protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The ¹⁹F-labeling method was 3.5-fold more sensitive than ¹⁵N-labeling, and could be combined with other chemical modification techniques such as lysine ¹³C-methylation. ¹³C-dimethylated-¹⁹F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.     

Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.     

Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.     

Adaptive mutation in Saccharomyces cerevisiae

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.     

Bone cancer risk in mice exposed to 224Ra: protraction effects from promotion

This paper analyzes data for the osteosarcoma incidence in life-time experiments of (224)Ra injected mice with respect to the importance of initiating and promoting action of ionizing high LET-radiation. This was done with the biologically motivated two step clonal expansion (TSCE) model of tumor induction. Experimentally derived osteosarcoma incidence in 1,194 mice following exposure to (224)Ra with different total radiation doses and different fractionation patterns were analyzed together with incidence data from 1,710 unirradiated control animals. Effects of radiation on the initiating event and on the clonal expansion rate, i.e. on promotion were found to be necessary to explain the observed patterns with this model. The data show a distinct inverse protraction effect at high doses, whereas at lower doses this effect becomes insignificant. Such a behavior is well reproduced in the proposed model: At dose rates above 6 mGy/day a longer exposure produces higher ERR per dose, while for lower rates the reverse is the case. The TSCE model permits the deduction of several kinetic parameters of a postulated two-step bone tumorigenesis process. Mean exposure rates of 0.13 mGy/day are found to double the baseline initiation rate. At rates above 100 mGy/day, the initiation rate decreases. The clonal expansion rate is doubled at 8 mGy/day, and it levels out at rates beyond 100 mGy/day.