Here, we present a new generation of nanoscale probes for in vivo monitoring of protease activity by fluorescence resonance energy transfer (FRET). The approach is based on a genetically programmable protein module carrying a fluorescently labeled, protease-specific sequence that can self-assemble onto quantum dots. The protein module was used for real-time detection of human immunodeficiency virus type-1 protease (HIV-1 Pr) activity as well as quantitative assessment of inhibitor efficiency.

In the absence of mechanical stimulation, brief exposure of osteoblasts to 1a,25(OH) 2 vitamin D 3 (1,25D) triggers plasma membrane electrical responses that couple to exocytosis. Here we describe for the first time 1,25D induction of exocytotic ATP release in static ROS 17/2.8 and SAOS-2 cells and primary calvarial osteoblasts expressing a vitamin D receptor (VDR). We found that 10 nM 1,25D optimally induced 45 ± 1% and 40 ± 1% of partial and complete exocytotic events, respectively, from a 1,25D-sensitive pool of ATP-containing secretory vesicles within 60 s. We measured a dose-dependent 1,25D induction of ATP secretion, with maximal response of ;6.2-fold (16.93 ± 1.82 nM for SAOS-2) and 3.1-fold (18.89 ± 1.39 nM for ROS 17/2.8) obtained with 10 nM 1,25D compared with basal ATP levels (2.75 ± 0.39 nM, SAOS-2; 6.09 ± 0.58 nM, ROS 17/2.8 cells). The natural metabolite 25(OH)vitamin D 3 (25D, 10 nM) induced a significant 3.6-fold increase of ATP release in ROS 17/2.8 cells, but there was no induction with the antagonist 1b,25(OH) 2 vitamin D 3 (1b,25D, 10 nM) or the steroid 17b-estradiol (10 nM). 1,25D-induced ATP secretion was abolished when cells were preincubated with inhibitors of vesicular exocytosis. siRNA VDR silencing prevented 1,25D stimulation of ATP exocytosis in ROS 17/2.8 and SAOS-2 cells. Similarly, 1,25D failed to stimulate ATP exocytosis in primary osteoblasts from a VDR knockout mouse. ATP secretion coupled to 1,25D induction of cytosolic calcium and chloride channel potentiation. Rapid 1,25D stimulation of ATP secretion involving nontranscriptional VDR functions in osteoblasts may help explain 1,25D bone anabolic properties.

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Abstract1α,25(OH) 2 -vitamin D 3 (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca 2+ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca 2+ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca 2+ -dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca 2+ -dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.

HIV protease plays a central role in its life cycle leading to release of functional viral particles. It has been successfully used as a therapeutic target to block HIV infection. Several protease inhibitors (PIs) are currently being employed as a part of anti-HIV therapy. However, the constant genetic drift in the virus leads to accumulation of mutations in both cleavage site and the protease, resulting in resistance and failure of therapy. We reported the use of a quantum dot (QD)-based protein probe for the in vivo monitoring of HIV-1 protease activity based on fluorescence resonance energy transfer. In the current study, we demonstrate the utility of this approach by quantifying the in vivo cleavage rates of three known protease and cleavage site mutations in the presence or absence of different PIs. The changes in IC50 values for the different PIs were similar to that observed in patients, validating our assay as a rapid platform for PI screening.