Xiaohui Wang

Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells

Accumulating evidence has shown that dysfunctional mitochondria can be selectively removed by mitophagy. Dysregulation of mitophagy is implicated in the development of neurodegenerative disease and metabolic disorders. How individual mitochondria are recognized for removal and how this process is regulated remain poorly understood. Here we report that FUNDC1, an integral mitochondrial outer-membrane protein, is a receptor for hypoxia-induced mitophagy. FUNDC1 interacted with LC3 through its typical LC3-binding motif Y(18)xxL(21), and mutation of the LC3-interaction region impaired its interaction with LC3 and the subsequent induction of mitophagy. Knockdown of endogenous FUNDC1 significantly prevented hypoxia-induced mitophagy, which could be reversed by the expression of wild-type FUNDC1, but not LC3-interaction-deficient FUNDC1 mutants. Mechanistic studies further revealed that hypoxia induced dephosphorylation of FUNDC1 and enhanced its interaction with LC3 for selective mitophagy. Our findings thus offer insights into mitochondrial quality control in mammalian cells.

Microwave assisted one-step green synthesis of cell-permeable multicolor photoluminescent carbon dots without surface passivation reagents

A facile, economic and green one-step microwave synthesis route towards photoluminescent carbon dots is proposed. The preparation requires a carbohydrate (glycerol, glycol, glucose, sucrose, etc.) and a tiny amount of an inorganic ion, and can finish in just a few minutes, no surface passivation reagent is needed. The carbon dots are biologically compatible and show favorable optical properties and have potential applications in biolabeling and bioimaging.

Label-free colorimetric detection of single nucleotide polymorphism by using single-walled carbon nanotube intrinsic peroxidase-like activity.

Single-walled carbon nanotubes (SWNTs) have been considered to be leading candidates for nanodevice applications and novel drug delivery. Intriguingly, recent studies have shown that SWNTs have catalytic activity even in the absence of catalytic factors. Since hydrogen peroxide is an important oxidizing agent in biological systems, the catalytic reaction of SWNTs with H2O2 has received much attention. The SWNT catalytic mechanism is much debated, but it has been suggested that it is related to trace amounts of metal catalyst in SWNTs. H2O2 is a major reactive oxygen species in living organisms, and its overproduction is implicated in the development of numerous inflammatory diseases, such as atherosclerosis, chronic obstructive pulmonary disease, and hepatitis. Furthermore, as a product of many enzyme-catalyzed reactions, H2O2 can act as an indicator to monitor the quantity of biologically important molecules, such as glucose. Therefore, studying the catalytic reaction of SWNTs with H2O2 might offer a promising application for disease diagnosis and for the design of SWNT-based sensors. In this work, we report that SWNTs possess intrinsic peroxidase-like activity. That the catalytic activity does not depend on trace amounts of metal catalyst in the SWNTs is evidenced by energy-dispersive X-ray (EDX) analysis. In the presence of H2O2, SWNTs catalyze the reaction of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) thereby producing a color change (Scheme 1). Our results indicate that the catalytic efficiency of SWNTs is strongly dependent on pH, temperature, and H2O2 concentration, similar to horseradish peroxidase (HRP). More importantly, we compared the catalytic efficiency of SWNTs containing different amounts of cobalt residues. The results clearly show that the observed “catalytic” effect of SWNTs can be attributed to their intrinsic properties rather than metal residues. Peroxidase activity has a great potential for practical application and has been used in the bioremediation of waste water or as diagnostic kits. As peroxidase mimics, SWNTs were used here for label-free colorimetric detection of disease-associated single-nucleotide polymorphism (SNP) with a direct detection limit of 1 nm based on the color reaction of TMB. It is well known that SNP detection is very important, and different kinds of detection methods have been reported; however, to our knowledge, this is the first demonstration of applying intrinsic SWNT peroxidase-like activity and color change for this purpose. This work will provide new insights into the utilization of SWNT peroxidase-like activity. To increase SWNT solubility in aqueous solution, we treated SWNTs with a mixture of concentrated sulfuric and nitric acids, as described previously. Figure S1 in the Supporting Information shows the mixed solution of H2O2 and TMB in the presence or absence of SWNTs. In the absence of SWNTs, the color of the solution does not change in 12 h; however, in the presence of SWNTs, the color changes from black to blue immediately. This result suggests that SWNTs can catalyze the reaction of TMB in the presence of [a] Y. Song, X. Wang, C. Zhao, K. Qu, Dr. J. Ren, Prof. X. Qu Laboratory of Chemical Biology Division of Biological Inorganic Chemistry State Key Laboratory of Rare Earth Resource Utilization Graduate School of the Chinese Academy of Sciences Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun, Jilin 130022 (China) Fax: (+86)431-85262656 E-mail : xqu@ciac.jl.cn Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.20090902643. Scheme 1. Schematic illustration of SWNTs catalyzing the reaction of peroxidase substrate TMB in the presence of H2O2 to give the blue product oxidized TMB (oxTMB).

A regulatory signaling loop comprising the PGAM5 phosphatase and CK2 controls receptor-mediated mitophagy.

Mitochondrial autophagy, or mitophagy, is a major mechanism involved in mitochondrial quality control via selectively removing damaged or unwanted mitochondria. Interactions between LC3 and mitophagy receptors such as FUNDC1, which harbors an LC3-interacting region (LIR), are essential for this selective process. However, how mitochondrial stresses are sensed to activate receptor-mediated mitophagy remains poorly defined. Here, we identify that the mitochondrially localized PGAM5 phosphatase interacts with and dephosphorylates FUNDC1 at serine 13 (Ser-13) upon hypoxia or carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) treatment. Dephosphorylation of FUNDC1 catalyzed by PGAM5 enhances its interaction with LC3, which is abrogated following knockdown of PGAM5 or the introduction of a cell-permeable unphosphorylated peptide encompassing the Ser-13 and LIR of FUNDC1. We further observed that CK2 phosphorylates FUNDC1 to reverse the effect of PGAM5 in mitophagy activation. Our results reveal a mechanistic signaling pathway linking mitochondria-damaging signals to the dephosphorylation of FUNDC1 by PGAM5, which ultimately induces mitophagy.

Opioid Activation of Toll-Like Receptor 4 Contributes to Drug Reinforcement

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4−/− mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

Chiral metallo-supramolecular complexes selectively recognize human telomeric G-quadruplex DNA

Here, we report the first example that one enantiomer of a supramolecular cylinder can selectively stabilize human telomeric G-quadruplex DNA. The P-enantiomer of this cylinder has a strong preference for G-quadruplex over duplex DNA and, in the presence of sodium, can convert G-quadruplexes from an antiparallel to a hybrid structure. The compounds chiral selectivity and its ability to discriminate quadruplex DNA have been studied by DNA melting, circular dichroism, gel electrophoresis, fluorescence spectroscopy and S1 nuclease cleavage. The chiral supramolecular complex has both small molecular chemical features and the large size of a zinc-finger-like DNA-binding motif. The complex is also convenient to synthesize and separate enantiomers. These results provide new insights into the development of chiral anticancer agents for targeting G-quadruplex DNA.

Gossypol induces Bax/Bak-independent activation of apoptosis and cytochrome c release via a conformational change in Bcl-2

Cells without Bak and Bax are largely resistant to apoptosis (1;2), despite the presence of other key components of the apoptotic machinery. We screened 7,800 natural compounds and found several that could specifically induce caspase activation and the release of cytochrome c (cyto c) in the bak//bax/ cells. One of these was gossypol, a polyphenolic compound naturally found in cottonseed that has been used in antifertility trials. We found that gossypol, but not other Bcl‐2‐interacting molecules, induced cyto c release and loss of mitochondrial membrane potential (m) independently of mPTP and Bak/Bax activation. Furthermore, we found that gossypol induced an allosteric change in Bcl‐2 in both bak//bax/ cells and Bcl‐2 overexpressing cells. This change in Bcl‐2 conformation led to the release of cyto c in the presence of Bcl‐2 and Bcl‐xL in reconstituted proteoliposomes. We also observed that gossypol substantially reduced the growth of tumor xenografts from Bcl‐2 overexpressing cells in nude mice. We conclude that gossypol converts the antiapoptotic molecule Bcl‐2 into a proapoptotic molecule that can mediate the release of cyto c and induce apoptosis—Lei, X., Chen, Y., Du, G., Yu, W., Wang, X., Qu, H., Xia, B., He, H., Mao, J., Zong, W., Liao, X., L., Mehrpour, M., Hao, X., Chen, Q. Gossypol induces Bax/Bak‐independent activation of apoptosis and cytochrome c release via a conformational change in Bcl‐2. FASEB J. 20, E1510 –E1519 (2006)

Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells

HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5′-LTR and 3′-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future.

i-Motif Quadruplex DNA-Based Biosensor for Distinguishing Single- and Multiwalled Carbon Nanotubes

The increasing worldwide demand for carbon nanotubes (CNTs) and increasing concern regarding how to safely develop and use CNTs are requiring a low-cost, simple, and highly sensitive CNT detection assay for toxicological evaluation and environmental monitoring. However, this goal is still far from being achieved. All the current CNT detection techniques are not applicable for automation and field analysis because they are dependent on highly expensive special instruments and complicated sample preparation. On the basis of the capability of single-walled carbon nanotubes (SWNTs) to specifically induce human telomeric i-motif formation, we design an electrochemical DNA (E-DNA) sensor that can distinguish single- and multiwalled carbon nanotubes both in buffer and in cell extracts. The E-DNA sensor can selectively detect SWNTs with a direct detection limit of 0.2 ppm and has been demonstrated in cancer cell extracts. To the best of our knowledge, this is the first demonstration of a biosensing technique that can distinguish different types of nanotubes. Our work will provide new insights into how to design a biosensor for detection of carbon nanotubes.

A small natural molecule promotes mitochondrial fusion through inhibition of the deubiquitinase USP30

Mitochondrial fusion is a highly coordinated process that mixes and unifies the mitochondrial compartment for normal mitochondrial functions and mitochondrial DNA inheritance. Dysregulated mitochondrial fusion causes mitochondrial fragmentation, abnormal mitochondrial physiology and inheritance, and has been causally linked with a number of neuronal diseases. Here, we identified a diterpenoid derivative 15-oxospiramilactone (S3) that potently induced mitochondrial fusion to restore the mitochondrial network and oxidative respiration in cells that are deficient in either Mfn1 or Mfn2. A mitochondria-localized deubiquitinase USP30 is a target of S3. The inhibition of USP30 by S3 leads to an increase of non-degradative ubiquitination of Mfn1/2, which enhances Mfn1 and Mfn2 activity and promotes mitochondrial fusion. Thus, through the use of an inhibitor of USP30, our study uncovers an unconventional function of non-degradative ubiquitination of Mfns in promoting mitochondrial fusion.