Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides.

Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

Zhaoshumycins A and B, Two Unprecedented Antimycin-Type Depsipeptides Produced by the Marine-Derived Streptomyces sp. ITBB-ZKa6.

Marine actinomycetes are prolific chemical sources of complex and novel natural products, providing an excellent chance for new drug discovery. The chemical investigation of the marine-derived Streptomyces sp. ITBB-ZKa6, from Zhaoshu island, Hainan, led to the discovery of two unique antimycin-type depsipeptides, zhaoshumycins A (1) and B (2), along with the isolation of the four known neoantimycins A (3), F (4), D (5), and E (6). The structures of the new compounds 1 and 2 were elucidated on the basis of the analysis of diverse spectroscopic data and biogenetic consideration. Zhaoshumycins A (1) and B (2) represent a new class of depsipeptides, featuring two neoantimycin monomers (only neoantimycin D or neoantimycins D and E) linked to a 1,4-disubstituted benzene ring via an imino group. Initial toxicity tests of 1-6 in MCF7 human breast cancer cells revealed that compounds 5 and 6 possess weak cytotoxic activity. Further structure-activity relationship analysis suggested the importance of the NH2 group at C-34 in 5 and 6 for cytotoxicity in MCF7 cells.

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.

A Standalone ß-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold.

Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone ß-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone ß-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone ß-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.

Inhibition of mitochondrial complex III induces differentiation in acute myeloid leukemia.

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.

Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease.

The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

Structure-activity-distribution relationship study of anti-cancer antimycin-type depsipeptides.

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage.

Mitochondrial impairment and calcium (Ca++) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca++-ATPase pumps are impaired causing cytoplasmic Ca++ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser65, and reduced calpain-cleavage of Parkin. Treatment with the Ca++ ionophore A23187, which facilitates Ca++ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca++ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca++-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.

The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine.

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca2+ from intracellular stores and activation of Ca2+-activated Cl- channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca2+ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na+/Ca2+ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca2+ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca2+ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca2+ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157 inhibited Ca2+ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca2+ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca2+ (CaV3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced CaV3.2 currents. CCCP blocked CaV3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca2+ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.

A phylogenetic and evolutionary analysis of antimycin biosynthesis.

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of antibiotics. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XL-related anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 73 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and a phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ- or IP-form, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ- and IP-form biosynthetic pathways.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Inhibition of Human Cancer Cell Growth by Analogues of Antimycin A.

In a recent study, several new derivatives of antimycin A (AMA) were produced by means of a novel transacylation reaction, and these were shown to mediate selective toxicity toward cultured A549 human lung epithelial adenocarcinoma cells, as compared with WI-38 normal human lung fibroblasts. The purpose of our study was to investigate whether the analogues all expressed their cytotoxicity by the same mechanism. This was done by studying the effects of the compounds in several types of cell lines. In comparison with 2-O-methylantimycin, which acts at the locus of Bcl-2, none of the new derivatives exhibited a difference in cytotoxicity toward cells expressing different levels of Bcl-2. In cell lines that over- or underexpress estrogen or Her2 receptors, AMA analogue 2 exhibited Her2 receptor dependency at low concentration. Three compounds (1, 4, and 6) exhibited concentration-dependent increases in reactive oxygen species, with 6 being especially potent. Compounds 5 and 6 diminished mitochondrial membrane potential more potently than AMA, and 1 also displayed enhanced activity relative to 2-4. Interestingly, only 1 and AMA displayed strong inhibition of the respiratory chain, as measured by monitoring NADH (reduced nicotinamide adenine dinucleotide) oxidase. Because four of the analogues have positively charged substituents, two of these (4 and 6) were studied to see whether the observed effects were due to much higher level of accumulation within the mitochondria. Their presence in the mitochondria was not dramatically enhanced. Neither of the two presently characterized mechanisms of cell killing by AMA can fully account for the observed results.

Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation.

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1+ with free fatty acid anions resulting in uncoupling; ii) SkQ1H2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Qi), involving the reduction of SkQ1 to SkQ1H2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (Jm) and cytosolic H2O2 (Jc) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of Jm and Jc but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished Jm established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H2 at Complex I IQ or Complex III Qi site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ10. Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Marine Streptomyces sp. derived antimycin analogues suppress HeLa cells via depletion HPV E6/E7 mediated by ROS-dependent ubiquitin-proteasome system.

Four new antimycin alkaloids (1-4) and six related known analogs (5-10) were isolated from the culture of a marine derived Streptomyces sp. THS-55, and their structures were elucidated by extensive spectroscopic analysis. All of the compounds exhibited potent cytotoxicity in vitro against HPV-transformed HeLa cell line. Among them, compounds 6-7 were derived as natural products for the first time, and compound 5 (NADA) showed the highest potency. NADA inhibited the proliferation, arrested cell cycle distribution, and triggered apoptosis in HeLa cancer cells. Our molecular mechanic studies revealed NADA degraded the levels of E6/E7 oncoproteins through ROS-mediated ubiquitin-dependent proteasome system activation. This is the first report that demonstrates antimycin alkaloids analogue induces the degradation of high-risk HPV E6/E7 oncoproteins and finally induces apoptosis in cervical cancer cells. The present work suggested that these analogues could serve as lead compounds for the development of HPV-infected cervical cancer therapeutic agents, as well as research tools for the study of E6/E7 functions.

Mitochondrial Nitroreductase Activity Enables Selective Imaging and Therapeutic Targeting.

Nitroreductase (NTR) activities have been known for decades, studied extensively in bacteria and also in systems as diverse as yeast, trypanosomes, and hypoxic tumors. The putative bacterial origin of mitochondria prompted us to explore the possible existence of NTR activity within this organelle and to probe its behavior in a cellular context. Presently, by using a profluorescent near-infrared (NIR) dye, we characterize the nature of NTR activity localized in mammalian cell mitochondria. Further, we demonstrate that this mitochondrially localized enzymatic activity can be exploited both for selective NIR imaging of mitochondria and for mitochondrial targeting by activating a mitochondrial poison specifically within that organelle. This constitutes a new mechanism for mitochondrial imaging and targeting. These findings represent the first use of mitochondrial enzyme activity to unmask agents for mitochondrial fluorescent imaging and therapy, which may prove to be more broadly applicable.

Hypoxia as a therapy for mitochondrial disease.

Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability. Genetic or small-molecule activation of the hypoxia response is protective against mitochondrial toxicity in cultured cells and zebrafish models. Chronic hypoxia leads to a marked improvement in survival, body weight, body temperature, behavior, neuropathology, and disease biomarkers in a genetic mouse model of Leigh syndrome, the most common pediatric manifestation of mitochondrial disease. Further preclinical studies are required to assess whether hypoxic exposure can be developed into a safe and effective treatment for human diseases associated with mitochondrial dysfunction.

Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes.

Differentiated podocytes, a type of renal glomerular cells, require substantial levels of energy to maintain glomerular physiology. Mitochondria and glycolysis are two major producers of ATP, but the precise roles of each in podocytes remain unknown. This study evaluated the roles of mitochondria and glycolysis in differentiated and differentiating podocytes. Mitochondria in differentiated podocytes are located in the central part of cell body while blocking mitochondria had minor effects on cell shape and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia, a cortical area of these cells, decreased the cell migratory ability and induced the apoptosis. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. In turn, synaptopodin expression was ameliorated by blocking either mitochondrial respiration or glycolysis. Similar to differentiated podocytes, the differentiating podocytes utilized the glycolysis for regulating apoptosis and lamellipodia formation while synaptopodin expression was likely involved in both mitochondrial OXPHOS and glycolysis. Finally, adult mouse podocytes have most of mitochondria predominantly in the center of the cytosol whereas phosphofructokinase, a rate limiting enzyme for glycolysis, was expressed in foot processes. These data suggest that mitochondria and glycolysis play parallel but distinct roles in differentiated and differentiating podocytes.