Antimycin

Antimycin A (more exactly Antimycin A1b) is a secondary metabolite produced by Streptomyces bacteria[1] and a member of a group of related compounds called antimycins. Antimycin A is classified as an extremely hazardous substance in the United States, as defined in Section 302 of the U.S. Emergency Planning and Community Right-to-Know Act (42 U.S.C. 11002), and is subject to strict reporting requirements by facilities which produce, store, or use it in significant quantities.[2]

antimycin

Antimycin A is an inhibitor of cellular respiration, specifically oxidative phosphorylation. Antimycin A binds to the Qi site of cytochrome c reductase, inhibiting the oxidation of ubiquinol to ubiquinone in the Qi site, thereby disrupting the Q-cycle of enzyme turn over. It also will cause the disruption of the entire electron transport chain. Due to this, there can be no production of ATP. Cytochrome c reductase is a central enzyme in the electron transport chain of oxidative phosphorylation.[4]The inhibition of this reaction disrupts the formation of the proton gradient across the inner membrane of the mitochondria. The production of ATP is subsequently inhibited, as protons are unable to flow through the ATP synthase complex in the absence of a proton gradient. This inhibition also results in the formation of the toxic free radical superoxide.[5] In presence of antimycin A the dependence of the superoxide production rate on oxygen level is hyperbolic.[6] In cultured cells at the background of mitochondrial respiration inhibition, the rate of superoxide production exceeds the cellular mechanisms to scavenge it, overwhelming the cell and leading to cell death.[citation needed]

Fungus-growing attine ants have been shown to use antimycins - produced by symbiotic Streptomyces bacteria - in their fungiculture, to inhibit non-cultivar (i.e. pathogenic) fungi.[9] One research group studying these symbiotic Streptomyces bacteria recently identified the biosynthetic gene cluster for antimycins, which was unknown despite the compounds themselves being identified 60 years ago. Antimycins are synthesised by a hybrid polyketide synthase (PKS)/non-ribosomal peptide synthase (NRPS).[10]

The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

Antimycin A is an inhibitor of Complex III (CIII). It binds to the Qi site of CIII and inhibits the transfer of electrons from heme bH to oxidized Q (Qi site inhibitor). High concentrations of antimycin A also inhibit acyl-CoA oxidase and D-amino acid oxidase.

A respiratory inhibitor, antimycin A (AA), induced an apoptotic-like cell death characterized by nuclear and DNA fragmentation in human leukemia HL-60 cells. This cell death was significantly restricted by a nitric oxide synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA), and a poly(ADP-ribose) polymerase (PARP) inhibitor, 5-aminoisoquinoline (AIQ). Indeed, NO production and PARP overactivation were detected in the cells treated with AA. On the one hand, L-NMMA partly eliminated NO production and on the other, AIQ and L-NMMA also restricted PARP activation. Excessive signals related to PARP overactivation induce the translocation of an apoptosis-inducing factor (AIF) from the mitochondria to the nuclei, resulting in DNA fragmentation. In AA-treated cells, the nuclear translocation of AIF occurred. This translocation was restricted by pretreatment with AIQ and L-NMMA. Although pretreatment with ascorbic acid eliminated the reactive oxygen species (ROS) generation induced by the blockade of complex III by AA, the pretreatment did not protect the cells from AA-induced cell death. Furthermore, cytochrome c release or caspase-3 activation was not observed in the cells treated with AA. These results suggest that AA-induced cell death does not depend on respiratory inhibition and the succeeding cascades, but on NO production, PARP overactivation and AIF translocation.

Acute changes in environmental parameters (e.g., O2, pH, UV, osmolarity, nutrients, etc.) evoke a common transcriptomic response in yeast referred to as the "environmental stress response" (ESR) or "common environmental response" (CER). Why such a diverse array of insults should elicit a common transcriptional response remains enigmatic. Previous functional analyses of the networks involved have found that, in addition to up-regulating those for mitigating the specific stressor, the majority appear to be involved in balancing energetic supply and demand and modulating progression through the cell cycle. Here we compared functional and regulatory aspects of the stress responses elicited by the acute inhibition of respiration with antimycin A and oxygen deprivation under catabolite non-repressed (galactose) conditions.

To further investigate the regulatory factors involved and distinguish between networks that respond to oxygen deprivation and those that respond to the abrupt cessation of respiration, we poisoned the respiratory chain with the cytochrome bc1 inhibitor antimycin A and conducted a temporal analysis of the transcriptomic response over three generations of aerobiosis followed by three generations of anaerobiosis. This treatment served several purposes. First, poisoning respiration under normoxic conditions mimics the loss of respiratory-dependent energy production that occurs during the shift to anaerobiosis, independent of a change in oxygen availability. Given that changes in the activity of more chronically responding gene networks are observed within three generations of anaerobic growth [6], assaying gene expression over this period should allow us to differentiate between networks that respond to the cessation of respiration and any that may respond to the chronic loss of respiratory capacity, independent of a change in oxygen availability. Moreover, by then shifting the respiratory incompetent cells to anaerobiosis and assaying gene expression over three additional generations of growth, we should be able to distinguish between any networks that acutely respond to the loss of oxygen and those that more chronically respond to oxygen-dependent changes in cellular heme concentrations, for example those that are regulated by Hap1, Hap2/3/4/5, Rox1, Upc2 and Mot3 [6]. Overall, we expected and found that the same ESR/CER gene networks respond to poisoning of the respiratory chain under normoxia as to the shift to anoxia in the absence of inhibitor, networks that appear to be involved in balancing energy supply and demand, growth rate, and progression through the cell cycle.

Comparison of Figure 1C with 1A shows the presence of antimycin A drastically alters the anaerobic response; the large numbers of transiently responding genes observed in Figure 1A are conspicuously absent, leaving only those that respond after a substantial delay (2 generations). Interestingly, the dynamics of this response is remarkably similar to that elicited by anoxia in catabolite-repressed cells [5, 6]. Taken together these results suggest that the acute, transient transcriptomic response observed in Figures 1A and 1B is evoked by the inhibition of respiration, whether by chemical means (antimycin A) under normoxic conditions (Figure 1B) or the rapid removal of oxygen from respiratory competent cells (Figure 1A), whereas the delayed, chronic phase (Figures 1A and 1C) is invoked by oxygen deprivation.

Comparison of genes that comprise the acute, transient phase of the anaerobic response with those that respond to antimycin A treatment in air and subsequent anaerobiosis. Genes that responded within the first 0.25 generations of anaerobiosis were classified as acutely responding (acute N2) whereas those that responded after this period were classified as chronically responding (chronic N2) (see Figure 1A for phase dynamics). Genes that comprise each of these classes are compared to those that responded to the antimycin A treatments (light gray for air and darker gray for N2). The area of the circles is scaled to the number of genes (ORFs), and the percentage of genes found in each phase is indicated in the parentheses.

This study has revealed novel insight into the transcriptomic response referred to as the "common environmental response" (CER) [4] or "environmental stress response" (ESR) [2] and differentiates between networks that respond to respiratory inhibition and those that respond to oxygen deprivation. First, we show that the acute inhibition of respiration by either oxygen deprivation (anoxia) or chemical means (antimycin A) in catabolite non-repressed conditions evokes a transient ESR/CER-like response. Gene network analyses suggest that changes in the activity of Fhl1 and PAC/RRPE-associated factors result in the transient down-regulation of genes involved in energetically costly programs of ribosomal biogenesis, protein synthesis, and rRNA transcription/processing. Simultaneously, transient changes in the activity of the SBF (Swi4-Swi6) and MBF complexes (Mbp1-Swi6) result in the down-regulation of genes involved in late G1 and the G1/S transition of the cell cycle and a predicted delay in its progression as mass and energy are assessed before committing to another round. At the same time Msn2/4-regulated networks involved in carbohydrate import/utilization and reserve energy metabolism are transiently activated. 041b061a72