Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations.

BackgroundLecithin:cholesterol acyltransferase (LCAT) is an enzyme that catalyzes the esterification of free cholesterol carried in high‐density lipoproteins (HDL). Esterification of cholesterol results in internalization of cholesteryl ester (CE) within the core of maturing HDLs. Of the HDL species, increased levels of CE‐rich HDLs are inversely correlated with the development of cardiovascular disease. Prior research has established that the activity of LCAT is influenced by numerous external physiological stimuli. Our lab has previously developed nanoparticles (HDL‐NP) that mimic natural HDLs with regard to size, shape, surface chemistry, and cholesterol binding function. Herein, we describe the application of HDL‐NP as specific activators of LCAT and biosensors for assaying LCAT activity.MethodsHDL‐NP were synthesized using 5 nm diameter gold nanoparticles (AuNP) as a scaffold for the assembly of a lipid bilayer containing a mixture of phosphotidylcholine (PC), phosphotidylethanolamine (PE), and apolipoportein A‐I (apoAI). ApoAI, found in natural HDL, serves as a cofactor for LCAT. 10% of total nanoparticle phospholipid content consisted of a PC lipid labeled with the fluorophore, TopFluor, thus forming TopFluor‐labeled HDL‐NP (TF‐NP). A subset of HDL‐NP was formulated without apoAI, having only the phospholipid bilayer (BL‐NP), to serve as a control to demonstrate specificity for LCAT. These nanoparticle groups were incubated for 24 hours in PBS with cholesterol, LCAT, or cholesterol and LCAT. In addition, the particles were incubated in dilute serum. An increase in the fluorescence intensity was used as a measure of LCAT activity, presumably, due to a reduced quenching effect of the core AuNP on the TopFluor‐labeled PC.ResultsTF‐NP and BL‐NP were incubated with cholesterol alone, LCAT alone, or a mixture of cholesterol and LCAT (Fig. A). A significant increase in fluorescent signal was observed for the TF‐NP incubated with cholesterol and LCAT over that of the TF‐NP alone (p=0.0000005) indicating activity of LCAT on TF‐NP. Interestingly, the presence of cholesterol alone with TF‐NP and BL‐NP results in a fluorescence response, independent of apoAI content. The importance of apoAI as a cofactor for promoting LCAT activity is apparent through comparing the fluorescent response of TF‐NP and BL‐NP where the apoAI‐containing TF‐NP exhibits significantly increased fluorescence over BL‐NP when incubated with both cholesterol and LCAT (p=0.00012). Next, TF‐NP and BL‐NP were incubated in samples of diluted serum for 24 hours (Fig. B), demonstrating a dose‐response whereby increased serum resulted in a stronger fluorescent response from the nanoparticles. Furthermore, this study further confirms the specificity of LCAT towards apoAI‐containing TF‐NP, where fluorescence intensity was significantly increased over BL‐NP groups for all serum dilutions.ConclusionThese results demonstrate the ability of fluorescent HDL‐NP to serve as biosensors for detecting LCAT activity, and that detection of LCAT activity directly from serum samples is possible using this technology. Future work will aim to better understand cholesterol and cholesteryl binding to HDL‐NP through the observed quenching and de‐quenching fluorescent response, and to apply this biosensor for the detection of LCAT activity changes in response to both acute and prolonged exercise, both previously demonstrated to induce changes in LCAT activity in human subjects.Support or Funding InformationAFRL FA8650‐15‐2‐5518