Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling

<div><p>Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed <i>in vivo</i>. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function.</p></div

Properties of NO Sensor HTDAF-2.

<p>(A) Fluorescence spectra of HTDAF-2. Fluorescence excitation and emission spectra of 10 nM HTDAF-2 in PBS (pH 7.4) before (dark red lines) and after (orange lines) the addition of 0.5 mM NO donor (DEA NONOate) at 25°C. Excitation spectrum recorded at an emission wavelength of 525 nm shows a maximum at 488 nm. Emission spectrum recorded at an excitation wavelength of 480 nm shows a maximum at 512 nm. (B) The fluorescence intensities of HTDAF-2 in the presence of different concentrations of NO donor (DEA NONOate) normalized to the initial value. (C) The fluorescence response of HTDAF-2 after the addition of 2 mM xanthine/20 mU xanthine oxidase, 0.5 mM H₂O₂, NO²⁻, NO³⁻, MAHMA NONOate, and DEA NONOate for 30 min in PBS solution. (D) The fluorescence response of HTDAF-2 to NO donor (DEA NONOate) at the indicated pH. Error bars represent the standard deviation (SD).</p

Structures and reaction chemistry of HTDAF-2DA.

<p>Structures and reaction chemistry of HTDAF-2DA.</p

Fluorescence detection of NO in subcellular organelles of HeLa and MCF-7 cells.

<p>Targeted localization of HTDAF-2 by conjugation to HaloTag proteins in living HeLa cells. Images present HeLa cells expressing HaloTag in the cytosol/nucleus (A), nucleus (B), membrane (C), and mitochondria (D), with the red fluorescent protein mCherry fused with the same signal peptides. Scale bar = 10 μm. (E) Direct in-gel fluorescence of control (1), nucleus-HaloTag (2), plasma membrane-HaloTag (3), cytosol-HaloTag (4), and mitochondria-HaloTag (5) in HeLa cells labeled with 5 μM HTDAF-2DA. (F) The fluorescence responses of 5 μM HTDAF-2DA targeted in the plasma membrane to various concentrations of NO donor (DEA NONOate) in HeLa cells. (G and H) Kinetics of fluorescence response of 5 μM HTDAF-2DA in different subcellular compartments of HeLa (G) and MCF-7 (H) cells upon the addition of NO donor (DEA NONOate). Error bars represent SD.</p

Measurement of endogenous NO production in activated macrophages by HTDAF-2DA and DAF-2DA.

<p>(A and B) NO detection in Raw 264.7 macrophages expressing HaloTag in the cytosol/nucleus (A) or nucleus (B) stained by HTDAF-2DA. (C) NO detection in Raw 264.7 macrophages stainedby DAF-2DA. For A-C, cells were prestimulated for 8 h with LPS (0.5 μg/ml) and IFN-γ (250 U/ml) with or without L-NAME (2 mM). Data were measured in pooled cells with microplate reader. Error bars represent SD.</p

A fine-tuning mechanism underlying self-control for autophagy: deSUMOylation of BECN1 by SENP3

The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction. Abbreviations: AL: autolysosome; AP: autophagosome; ATG: autophagy related; ATG14: autophagy related 14; BECN1: beclin 1, autophagy related; cKO: conditional knockout; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle’s balanced salt solution; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PTM: post-translational modification; RFP: red fluorescent protein; ROS: reactive oxygen species; RUBCN/rubicon: RUN domain and cysteine-rich domain containing, BECN1-interacting protein; SENP3: SUMO specific peptidase 3; shRNA: small hairpin RNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SUMO: small ubiquitin-like modifier; UVRAG: UV radiation resistance associated gene.</p

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-1β) and anti-inflammatory cytokines (IL-10 and TGF-β), 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

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

DataSheet_1_Association of Human Whole Blood NAD+ Contents With Aging.docx

BackgroundNAD+, nicotinamide adenine dinucleotide, is mostly described to associate with the aging process. We aimed to investigate the association between human whole blood NAD+ contents and aging in a relative large-scale community-based population and further to address the gender impact on this association.MethodsWe recruited 1,518 participants aged over 18 years old and free of cardiovascular and any type of cancer from the Jidong community from 2019 to 2020. Whole blood NAD+ level was measured by cycling assay and LC-mass spectroscopy assay. The chronological age and clinical data were collected using standard questionnaires. The participants were divided into five groups according to their chronological age. General liner regression model was performed to analyze the association between NAD+ contents and aging. In addition, we also conducted subgroup analysis by gender.ResultsThe mean age of included 1,518 participants was 43.0 years, and 52.6% of them were men. The average levels of whole blood NAD+ of total participants was 33.0 ± 5.5 μmol/L. The whole blood NAD+ contents in men were significantly higher than that in women (34.5 vs. 31.3 μmol/L). There was significant difference in the meat diet among NAD+ quartile groups (p = 0.01). We observed a decline trend of NAD+ contents with aging before 50 years in total participants with significant level in 40–49 years old group (β coefficients with 95% confidence interval (95% CI): −1.12 (−2.18, −0.06)), while this trend disappeared after the 50 years. In addition, this association was significantly altered by gender (p for interaction = 0.003). In men, as compared with ≤29 years group, adjusted β coefficient decreased with aging but was only significant in the ≥60 year group (β,−2.16; 95% CI, −4.16 to −0.15). In females, the level of whole blood NAD+ did not significantly differ among five age groups and without the trend as males.ConclusionsAssociation of whole blood NAD+ contents with aging significantly differed in males and females. The loss of blood NAD+ with aging only was observed in males, especially in the male middle-aged population. It is crucial to consider the gender difference in further NAD+ related studies in the future.</p