Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA.

Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

Redox metabolism maintains the leukemogenic capacity and drug resistance of AML cells.

Rewiring of redox metabolism has a profound impact on tumor development, but how the cellular heterogeneity of redox balance affects leukemogenesis remains unknown. To precisely characterize the dynamic change in redox metabolism in vivo, we developed a bright genetically encoded biosensor for H2O2 (named HyPerion) and tracked the redox state of leukemic cells in situ in a transgenic sensor mouse. A H2O2-low (HyPerion-low) subset of acute myeloid leukemia (AML) cells was enriched with leukemia-initiating cells, which were endowed with high colony-forming ability, potent drug resistance, endosteal rather than vascular localization, and short survival. Significantly high expression of malic enzymes, including ME1/3, accounted for nicotinamide adenine dinucleotide phosphate (NADPH) production and the subsequent low abundance of H2O2. Deletion of malic enzymes decreased the population size of leukemia-initiating cells and impaired their leukemogenic capacity and drug resistance. In summary, by establishing an in vivo redox monitoring tool at single-cell resolution, this work reveals a critical role of redox metabolism in leukemogenesis and a potential therapeutic target.

NADPHnet: a novel strategy to predict compounds for regulation of NADPH metabolism via network-based methods.

Identification of compounds to modulate NADPH metabolism is crucial for understanding complex diseases and developing effective therapies. However, the complex nature of NADPH metabolism poses challenges in achieving this goal. In this study, we proposed a novel strategy named NADPHnet to predict key proteins and drug-target interactions related to NADPH metabolism via network-based methods. Different from traditional approaches only focusing on one single protein, NADPHnet could screen compounds to modulate NADPH metabolism from a comprehensive view. Specifically, NADPHnet identified key proteins involved in regulation of NADPH metabolism using network-based methods, and characterized the impact of natural products on NADPH metabolism using a combined score, NADPH-Score. NADPHnet demonstrated a broader applicability domain and improved accuracy in the external validation set. This approach was further employed along with molecular docking to identify 27 compounds from a natural product library, 6 of which exhibited concentration-dependent changes of cellular NADPH level within 100 µM, with Oxyberberine showing promising effects even at 10 µM. Mechanistic and pathological analyses of Oxyberberine suggest potential novel mechanisms to affect diabetes and cancer. Overall, NADPHnet offers a promising method for prediction of NADPH metabolism modulation and advances drug discovery for complex diseases.

Live-cell imaging reveals redox metabolic reprogramming during zygotic genome activation.

Metabolic programming is deeply intertwined with early embryonic development including zygotic genome activation (ZGA), the polarization of zygotic cells, and cell fate commitment. It is crucial to establish a noninvasive imaging technology that spatiotemporally illuminates the cellular metabolism pathways in embryos to track developmental metabolism in situ. In this study, we used two high-quality genetically encoded fluorescent biosensors, SoNar for NADH/NAD+ and iNap1 for NADPH, to characterize the dynamic regulation of energy metabolism and redox homeostasis during early zygotic cleavage. Our imaging results showed that NADH/NAD+ levels decreased from the early to the late two-cell stage, whereas the levels of the reducing equivalent NADPH increased. Mechanistically, transcriptome profiling suggested that during the two-cell stage, zygotic cells downregulated the expression of genes involved in glucose uptake and glycolysis, and upregulated the expression of genes for pyruvate metabolism in mitochondria and oxidative phosphorylation, with a decline in the expression of two peroxiredoxin genes, Prdx1 and Prdx2. Collectively, with the establishment of in situ metabolic monitoring technology, our study revealed the programming of redox metabolism during ZGA.

Association between NAD+ levels and anaemia among women in community-based study.

Nicotinamide adenine dinucleotide (NAD+ ) level is the protective factor of cardiovascular diseases (CVDs). In addition, anaemia is a risk factor of adverse cardiovascular outcomes in women. However, there are limited data about the association between NAD+ and anaemia. The aim of this study was to evaluate association of NAD+ with anaemia among women. A total of 727 females from Jidong community were included in the current analysis. NAD+ levels were tested by the cycling assay and HPLC assay using whole blood samples. Anaemia was determined by haemoglobin (Hb) concentration, and the subtypes of anaemia were further defined according to mean corpuscular volume (MCV) in blood. Multivariable logistic analysis was used to analyse the association between NAD+ levels and anaemia or its subtypes. The mean age of recruited subjects was 42.7 years. The proportion of anaemia by NAD+ levels quartiles were 19.7% (35/178), 4.8% (9/189), 3.4% (6/178) and 2.7% (5/182). Haematological parameters including haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and red blood count (RBC) increased over NAD+ quartiles. Red cell volume distribution width (RDW) decreased over NAD+ quartiles. Compared with the lowest quartile of NAD+ levels (<27.6µM), the adjusted odds ratios with 95% confidence intervals of the top quartile were 0.15 (0.06-0.41) for anaemia, 0.05 (0.01-0.36) for microcytic anaemia and 0.37 (0.10-1.36) for normocytic anaemia respectively. Higher NAD+ levels were significantly associated with lower prevalence of anaemia among women, especially microcytic anaemia and normocytic anaemia. Haematological parameters might serve as a predictor of the blood NAD+ levels.

MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells.

The connections between energy metabolism and stemness of hematopoietic stem cells (HSCs) at different developmental stages remain largely unknown. We generated a transgenic mouse line for the genetically encoded NADH/NAD+ sensor (SoNar) and demonstrate that there are 3 distinct fetal liver hematopoietic cell populations according to the ratios of SoNar fluorescence. SoNar-low cells had an enhanced level of mitochondrial respiration but a glycolytic level similar to that of SoNar-high cells. Interestingly, 10% of SoNar-low cells were enriched for 65% of total immunophenotypic fetal liver HSCs (FL-HSCs) and contained approximately fivefold more functional HSCs than their SoNar-high counterparts. SoNar was able to monitor sensitively the dynamic changes of energy metabolism in HSCs both in vitro and in vivo. Mechanistically, STAT3 transactivated MDH1 to sustain the malate-aspartate NADH shuttle activity and HSC self-renewal and differentiation. We reveal an unexpected metabolic program of FL-HSCs and provide a powerful genetic tool for metabolic studies of HSCs or other types of stem cells.

XA pH-Responsive and Colitis-Targeted Nanoparticle Loaded with Shikonin for the Oral Treatment of Inflammatory Bowel Disease in Mice.

Epidemiology shows that more than 6.8 million people in the world are influenced by inflammatory bowel disease (IBD) each year. IBD is a refractory inflammatory disease, and the disease mainly affects the colon. Shikonin (SK) was originally extracted from traditional Chinese medicine "Zicao" (with an English name Lithospermum erythrorhizon) and found to inhibit inflammation, regulate immunity, and be involved in healing wounds. Herein, we used chitosan (CS), hyaluronic acid (HA), and pH-responsive polymer Eudragits S100 (ES100) to design SK-loaded ES100/HA/CS nanoparticles (SK@SAC) as an oral delivery system to treat the colitis mice. Particle size of SK@SAC was 190.3 nm and drug loading efficiency was 6.6%. SAC nanoparticles accumulated in RAW264.7 macrophages and exhibited colitis-targeted ability by increasing the local drug concentration as well as reducing nonspecific distribution after oral gavage. In TNBS-induced IBD mice, SK@SAC treatment had significant therapeutic effects, regulated of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and anti-inflammatory cytokines (IL-10 and TGF-ß), and also inhibited COX-2 and iNOS activity. SK@SAC also increased tight junction protein ZO-1 and occludin to some extent. These promising results showed that this novel oral SK-loaded nanoparticle drug delivery system for targeted treatment provides a new strategy for the management of IBD.

A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells.

Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.

Fine-Tuning of PGC1α Expression Regulates Cardiac Function and Longevity.

RATIONALE: PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1α) represents an attractive target interfering bioenergetics and mitochondrial homeostasis, yet multiple attempts have failed to upregulate PGC1α expression as a therapy, for instance, causing cardiomyopathy. OBJECTIVE: To determine whether a fine-tuning of PGC1α expression is essential for cardiac homeostasis in a context-dependent manner. METHODS AND RESULTS: Moderate cardiac-specific PGC1α overexpression through a ROSA26 locus knock-in strategy was utilized in WT (wild type) mice and in G3Terc-/- (third generation of telomerase deficient; hereafter as G3) mouse model, respectively. Ultrastructure, mitochondrial stress, echocardiographic, and a variety of biological approaches were applied to assess mitochondrial physiology and cardiac function. While WT mice showed a relatively consistent PGC1α expression from 3 to 12 months old, age-matched G3 mice exhibited declined PGC1α expression and compromised mitochondrial function. Cardiac-specific overexpression of PGC1α (PGC1αOE) promoted mitochondrial and cardiac function in 3-month-old WT mice but accelerated cardiac aging and significantly shortened life span in 12-month-old WT mice because of increased mitochondrial damage and reactive oxygen species insult. In contrast, cardiac-specific PGC1α knock in in G3 (G3 PGC1αOE) mice restored mitochondrial homeostasis and attenuated senescence-associated secretory phenotypes, thereby preserving cardiac performance with age and extending health span. Mechanistically, age-dependent defect in mitophagy is associated with accumulation of damaged mitochondria that leads to cardiac impairment and premature death in 12-month-old WT PGC1αOE mice. In the context of telomere dysfunction, PGC1α induction replenished energy supply through restoring the compromised mitochondrial biogenesis and thus is beneficial to old G3 heart. CONCLUSIONS: Fine-tuning the expression of PGC1α is crucial for the cardiac homeostasis because the balance between mitochondrial biogenesis and clearance is vital for regulating mitochondrial function and homeostasis. These results reinforce the importance of carefully evaluating the PGC1α-boosting strategies in a context-dependent manner to facilitate clinical translation of novel cardioprotective therapies.

Discovery of Natural Products Targeting NQO1 via an Approach Combining Network-Based Inference and Identification of Privileged Substructures.

NAD(P)H:quinone oxidoreductase 1 (NQO1) has been shown to be a potential therapeutic target for various human diseases, such as cancer and neurodegenerative disorders. Recent advances in computational methods, especially network-based methods, have made it possible to identify novel compounds for a target with high efficiency and low cost. In this study, we designed a workflow combining network-based methods and identification of privileged substructures to discover new compounds targeting NQO1 from a natural product library. According to the prediction results, we purchased 56 compounds for experimental validation. Without the assistance of privileged substructures, 31 compounds (31/56 = 55.4%) showed IC50 < 100 µM, and 11 compounds (11/56 = 19.6%) showed IC50 < 10 µM. With the assistance of privileged substructures, the two success rates were increased to 61.8 and 26.5%, respectively. Seven natural products further showed inhibitory activity on NQO1 at the cellular level, which may serve as lead compounds for further development. Moreover, network analysis revealed that osthole may exert anticancer effects against multiple cancer types by inhibiting not only carbonic anhydrases IX and XII but also NQO1. Our workflow and computational methods can be easily applied in other targets and become useful tools in drug discovery and development.

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.

Blakeslea trispora Photoreceptors: Identification and Functional Analysis.

Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trisporaIMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

A Negative Regulator of Carotenogenesis in Blakeslea trispora.

As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of ß-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more ß-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids.IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

Safe and Efficacious Diphtheria Toxin-Based Treatment for Melanoma: Combination of a Light-On Gene-Expression System and Nanotechnology.

The controversy surrounding the use of diphtheria toxin (DT) as a therapeutic agent against tumor cells arises mainly from its unexpected harmfulness to healthy tissues. We encoded the cytotoxic fragment A of DT (DTA) as an objective gene in the Light-On gene-expression system to construct plasmids pGAVPO (pG) and pU5-DTA (pDTA). Meanwhile, a cRGD-modified ternary complex comprising plasmids, chitosan, and liposome (pG&pDTA@cRGD-CL) was prepared as a nanocarrier to ensure transfection efficiency. Benefiting from spatiotemporal control of this light-switchable transgene system and the superior tumor targeting of the carrier, toxins were designed to be expressed selectively in illuminated lesions. In vitro studies suggested that pG&pDTA@cRGD-CL exerted arrest of the S phase in B16F10 cells upon blue light irradiation and, ultimately, induced the apoptosis and necrosis of tumor cells. Such DTA-based treatment exerted enhanced antitumor activity in mice bearing B16F10 xenografts and displayed prolonged survival time with minimal side effects. Hence, we described novel DTA-based therapy combined with nanotechnology and the Light-On gene-expression system: such treatment could be a promising strategy against melanoma.

SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer.

Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid ß-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.

SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth.

The malate-aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate-aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD(+) redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate-aspartate NADH shuttle activity and oxidative protection.

Imaging the Redox States of Live Cells with the Time-Resolved Fluorescence of Genetically Encoded Biosensors.

Redox environments in cells influence many important physiological and pathological processes. In this study, the time-resolved fluorescence of a recently reported thiol redox-sensitive sensor based on vertebrate fluorescent protein UnaG, roUnaG, was studied, along with the application of the time-resolved fluorescence of roUnaG to image the redox states of the mitochondria, cytoplasm, and nucleus in live cells. Time-resolved fluorescence images of roUnaG clearly demonstrated that potent anticancer compound KP372-1 induced extreme oxidative stress. A more stressful redox state observed in activated macrophages further demonstrated the validity of roUnaG with time-resolved fluorescence. For comparison, time-resolved fluorescence images of four other frequently used redox biosensors (roGFP1, HyPer, HyPerRed, and rxRFP) were also captured. The time-resolved fluorescence allows an intrinsically ratiometric measurement for biosensors with one excitation wavelength and provides new opportunities for bioimaging.

Ultrafast internal conversion dynamics of bilirubin bound to UnaG and its N57A mutant.

Fluorescent proteins (FPs) have become fundamental tools for live cell imaging. Most FPs currently used are members of the green fluorescent protein super-family, but new fluorophores such as bilin-FPs are being developed and optimized. In particular, the UnaG FP incorporates bilirubin (BR) as a chromophore, enhancing its fluorescence quantum yield by three orders of magnitude relative to that in solution. To investigate the mechanism of this dramatic enhancement and provide a basis for further engineering of UnaG and other tetrapyrrole-based fluorophores, we performed picosecond fluorescence and femtosecond transient absorption measurements of BR bound to UnaG and its N57A site-directed mutant. The dynamics of wt-UnaG, which has a fluorescence QY of 0.51, are largely homogeneous, showing an excited state relaxation of ∼200 ps, and a 2.2 ns excited-state lifetime decay with a kinetic isotope effect (KIE) of 1.1 for D2O vs. H2O buffer. In contrast, for UnaG N57A (fluorescence QY 0.01) the results show a large spectral inhomogeneity with excited state decay timescales of 47 and 200 ps and a KIE of 1.4. The non-radiative deactivation of the excited state is limited by proton transfer. The loss of direct hydrogen bonds to the endo-vinyl dipyrrinone moiety of BR leads to high flexibility and structural heterogeneity of UnaG N57A, as seen in the X-ray crystal structure.

Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re-expression of wild-type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2-dependent manner. The SIRT2-mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.