Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.

Hepatocellular carcinoma (HCC) is a common cause of cancer-related death worldwide. As obesity and diabetes become more prevalent, the contribution of non-alcoholic fatty liver disease (NAFLD) to HCC is rising. Recently, we reported intrahepatic CD4+ T cells are critical for anti-tumor surveillance in NAFLD. Lipid accumulation in the liver is the hallmark of NAFLD, which may perturb T cell function. We sought to investigate how the lipid-rich liver environment influences CD4+ T cells by focusing on carnitine palmitoyltransferase (CPT) family members, which control the mitochondrial β-oxidation of fatty acids and act as key molecules in lipid catabolism. Linoleic acid (C18:2) co-localized within the mitochondria along with a corresponding increase in CPT gene upregulation. This CPT upregulation can be recapitulated by feeding mice with a high-C18:2 diet or the NAFLD promoting methionine-choline-deficient (MCD) diet. Using an agonist and antagonist, the induction of CPT genes was found to be mediated by peroxisome proliferator-activated receptor alpha (PPAR-α). CPT gene upregulation increased mitochondrial reactive oxygen species (ROS) and led to cell apoptosis. In vivo, using liver-specific inducible MYC transgenic mice fed MCD diet, blocking CPT with the pharmacological inhibitor perhexiline decreased apoptosis of intrahepatic CD4+ T cells and inhibited HCC tumor formation. These results provide useful information for potentially targeting the CPT family to rescue intrahepatic CD4+ T cells and to aid immunotherapy for NAFLD-promoted HCC.