RESUMO
Hyperpolarized 13C-labeled fumarate probes tissue necrosis via the production of 13C-malate. Despite its promises in detecting tumor necrosis and kidney injuries, its clinical translation has been limited, primarily due to the low solubility in conventional glassing solvents. In this study, we introduce a new formulation of fumarate for dissolution dynamic nuclear polarization (DNP) by using meglumine as a counterion, a nonmetabolizable derivative of sorbitol. We have found that meglumine fumarate vitrifies by itself with enhanced water solubility (4.8 M), which is expected to overcome the solubility-restricted maximum concentration of hyperpolarized fumarate after dissolution. The achievable liquid-state polarization level of meglumine-fumarate is more than doubled (29.4 ± 1.3%) as compared to conventional dimethyl sulfoxide (DMSO)-mixed fumarate (13.5 ± 2.4%). In vivo comparison of DMSO- and meglumine-prepared 50-mM hyperpolarized [1,4-13C2]fumarate shows that the signal sensitivity in rat kidneys increases by 10-fold. As a result, [1,4-13C2]aspartate and [13C]bicarbonate in addition to [1,4-13C2]malate can be detected in healthy rat kidneys in vivo using hyperpolarized meglumine [1,4-13C2]fumarate. In particular, the appearance of [13C]bicarbonate indicates that hyperpolarized meglumine [1,4-13C2]fumarate can be used to investigate phosphoenolpyruvate carboxykinase, a key regulatory enzyme in gluconeogenesis.
Assuntos
Isótopos de Carbono , Fumaratos , Rim , Solubilidade , Animais , Fumaratos/química , Fumaratos/metabolismo , Ratos , Rim/metabolismo , Isótopos de Carbono/química , Gluconeogênese , Masculino , Ratos Sprague-DawleyRESUMO
The feasibility of hyperpolarized [2-13C, 3-2H3]pyruvate for probing gluconeogenesis in vivo was investigated in this study. Whereas hyperpolarized [1-13C]pyruvate has clear access to metabolic pathways that convert pyruvate to lactate, alanine, and bicarbonate, its utility for assessing pyruvate carboxylation and gluconeogenesis has been limited by technical challenges, including spectral overlap and an obscure enzymatic step that decarboxylates the labeled carbon. To achieve unambiguous detection of gluconeogenic products, the carbonyl carbon in pyruvate was labeled with 13C. To prolong the T1 relaxation time, [2-13C, 3-2H3]pyruvate was synthesized and dissolved with D2O after dynamic nuclear polarization. The T1 of [2-13C, 3-2H3]pyruvate in D2O could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2-13C]pyruvate in water. Hyperpolarized [2-13C, 3-2H3]pyruvate with D2O dissolution was applied to rat livers in vivo under normal feeding and fasting conditions. A gluconeogenic product, [2-13C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2-13C, 3-2H3]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vivo.
Assuntos
Gluconeogênese , Fígado , Ácido Pirúvico , Animais , Fígado/metabolismo , Fígado/química , Ácido Pirúvico/metabolismo , Ácido Pirúvico/química , Ratos , Masculino , Ratos Sprague-Dawley , Isótopos de Carbono/químicaRESUMO
Parahydrogen-induced polarization (PHIP) followed by polarization transfer to 13 C is a rapidly developing technique for the generation of 13 C-hyperpolarized substrates. Chirality plays an essential role in living systems and differential metabolism of enantiomeric pairs of metabolic substrates is well documented. Inspired by asymmetric hydrogenation, here we report stereoPHIP, which involves the addition of parahydrogen to a prochiral substrate with a chiral catalyst followed by polarization transfer to 13 C spins. We demonstrate that parahydrogen could be rapidly added to the prochiral precursor to both enantiomers of lactic acid (D and L), with both the (R,R) and (S,S) enantiomers of a chiral rhodium(I) catalyst to afford highly 13 C-hyperpolarized (over 20 %) L- and D-lactate ester derivatives, respectively, with excellent stereoselectivity. We also show that the hyperpolarized 1 H signal decays obtained with the (R,R) and (S,S) catalysts were markedly different. StereoPHIP expands the scope of conventional PHIP to the production of 13 C hyperpolarized chiral substrates with high stereoselectivity.
RESUMO
The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Nucleossomos , Transferência Ressonante de Energia de Fluorescência/métodos , Histonas/metabolismo , Cromatina/genética , Metilação de DNARESUMO
Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semisynthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.
Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Transcrição Gênica , RNA Polimerase II/química , UbiquitinaçãoRESUMO
Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semi-synthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3 K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA Polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.
RESUMO
Previously, we noted that carboxylated multi-walled carbon nanotubes (cMWNTs) coated with Pluronic® F-108 (PF108) bound to and were accumulated by macrophages, but that pristine multi-walled carbon nanotubes (pMWNTs) coated with PF108 were not (Wang et al., Nanotoxicology2018, 12, 677). Subsequent studies with Chinese hamster ovary (CHO) cells that overexpressed scavenger receptor A1 (SR-A1) and with macrophages derived from mice knocked out for SR-A1 provided evidence that SR-A1 was a receptor of PF108-cMWNTs (Wang et al., Nanomaterials (Basel) 2020, 10, 2417). Herein, we replaced the PF108 coat with bovine serum albumin (BSA) to investigate how a BSA corona affected the interaction of multi-walled carbon nanotubes (MWNTs) with cells. Both BSA-coated cMWNTs and pMWNTs bound to and were accumulated by RAW 264.7 macrophages, although the cells bound two times more BSA-coated cMWNT than pMWNTs. RAW 264.7 cells that were deleted for SR-A1 using CRISPR-Cas9 technology had markedly reduced binding and accumulation of both BSA-coated cMWNTs and pMWNTs, suggesting that SR-A1 was responsible for the uptake of both MWNT types. Moreover, CHO cells that ectopically expressed SR-A1 accumulated both MWNT types, whereas wild-type CHO cells did not. One model to explain these results is that SR-A1 can interact with two structural features of BSA-coated cMWNTs, one inherent to the oxidized nanotubes (such as COOH and other oxidized groups) and the other provided by the BSA corona; whereas SR-A1 only interacts with the BSA corona of BSA-pMWNTs.
RESUMO
The nucleosome is the basic packing unit of the eukaryotic genome. Dynamic interactions between DNA and histones in the nucleosome are the molecular basis of gene accessibility regulation that governs the kinetics of various DNA-templated processes such as transcription elongation by RNA Polymerase II (Pol II). On the basis of single-molecule FRET measurements with chemically modified histones, we investigated the nucleosome dynamics during transcription elongation and how it is affected by histone acetylation at H3 K56 and the histone chaperone Nap1, both of which can affect DNA-histone interactions. We observed that H3K56 acetylation dramatically shortens the pause duration of Pol II near the entry region of the nucleosome, while Nap1 induces no noticeable difference. We also found that the elongation rate of Pol II through the nucleosome is unaffected by the acetylation or Nap1. These results indicate that H3K56 acetylation facilitates Pol II translocation through the nucleosome by assisting paused Pol II to resume and that Nap1 does not affect Pol II progression. Following transcription, only a small fraction of nucleosomes remain intact, which is unaffected by H3K56 acetylation or Nap1. These results suggest that (i) spontaneous nucleosome opening enables Pol II progression, (ii) Pol II mediates nucleosome reassembly very inefficiently, and (iii) Nap1 in the absence of other factors does not promote nucleosome disassembly or reassembly during transcription.
Assuntos
DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica , Acetilação , Sequência de Bases , DNA Polimerase II/metabolismo , Transferência Ressonante de Energia de Fluorescência , tRNA Metiltransferases/metabolismoRESUMO
The biological response of multi-walled carbon nanotubes (MWNTs) is related to their physicochemical properties and a thorough MWNT characterization should accompany an assessment of their biological activity, including their potential toxicity. Beyond characterizing the physicochemical properties of MWNTs from different sources or manufacturers, it is also important to characterize different production lots of the same MWNT product from the same vendor (i.e., lot-to-lot batch consistency). Herein, we present a comprehensive physicochemical characterization of two lots of commercial pristine MWNTs (pMWNTs) and carboxylated MWNTs (cMWNTs) used to study the response of mammalian macrophages to MWNTs. There were many similarities between the physicochemical properties of the two lots of cMWNTs and neither significantly diminished the 24-h proliferation of RAW 264.7 macrophages up to the highest concentration tested (200 µg cMWNTs/mL). Conversely, several physicochemical properties of the two lots of pMWNTs were different; notably, the newer lot of pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the original lot. Furthermore, a 72-h half maximal inhibitory concentration (IC-50) of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with the new lot of pMWNTs. These results demonstrate that subtle physicochemical differences can lead to significantly dissimilar cellular responses, and that production-lot consistency must be considered when assessing the toxicity of MWNTs.
RESUMO
The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αß TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.