Publications by authors named "Fedor Severin"

50 Publications

Lipophilic Cations Rescue the Growth of Yeast under the Conditions of Glycolysis Overflow.

Biomolecules 2020 09 20;10(9). Epub 2020 Sep 20.

Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 1-40 Leninskie Gory, 119991 Moscow, Russia.

Chemicals inducing a mild decrease in the ATP/ADP ratio are considered as caloric restriction mimetics as well as treatments against obesity. Screening for such chemicals in animal model systems requires a lot of time and labor. Here, we present a system for the rapid screening of non-toxic substances causing such a de-energization of cells. We looked for chemicals allowing the growth of yeast lacking trehalose phosphate synthase on a non-fermentable carbon source in the presence of glucose. Under such conditions, the cells cannot grow because the cellular phosphate is mostly being used to phosphorylate the sugars in upper glycolysis, while the biosynthesis of bisphosphoglycerate is blocked. We reasoned that by decreasing the ATP/ADP ratio, one might prevent the phosphorylation of the sugars and also boost bisphosphoglycerate synthesis by providing the substrate, i.e., inorganic phosphate. We confirmed that a complete inhibition of oxidative phosphorylation alleviates the block. As our system includes a non-fermentable carbon source, only the chemicals that did not cause a complete block of mitochondrial ATP synthesis allowed the initial depletion of glucose followed by respiratory growth. Using this system, we found two novel compounds, dodecylmethyl diphenylamine (FS1) and diethyl (tetradecyl) phenyl ammonium bromide (Kor105), which possess a mild membrane-depolarizing activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10091345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563754PMC
September 2020

Protonophore FCCP provides fitness advantage to PDR-deficient yeast cells.

J Bioenerg Biomembr 2020 10 17;52(5):383-395. Epub 2020 Aug 17.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskiye Gory 1-40, Moscow, 119991, Russia.

Pleiotropic drug resistance (PDR) plasma membrane transporters mediate xenobiotic efflux from the cells and thereby help pathogenic microorganisms to withstand antimicrobial therapies. Given that xenobiotic efflux is an energy-consuming process, cells with upregulated PDR can be sensitive to perturbations in cellular energetics. Protonophores dissipate proton gradient across the cellular membranes and thus increase ATP spendings to their maintenance. We hypothesised that chronic exposure of yeast cells to the protonophores can favour the selection of cells with inactive PDR. To test this, we measured growth rates of the wild type Saccharomyces cerevisiae and PDR-deficient Δpdr1Δpdr3 strains in the presence of protonophores carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), pentachlorophenol (PCP) and niclosamide (NCA). Although the protonophore-induced respiration rates of these two strains were similar, the PDR-deficient strain outperformed the control one in the growth rate on non-fermentable carbon source supplemented with low concentrations of FCCP. Thus, active PDR can be deleterious under conditions of partially uncoupled oxidative-phosphorylation. Furthermore, our results suggest that tested anionic protonophores are poor substrates of PDR-transporters. At the same time, protonophores imparted azole tolerance to yeasts, pointing that they are potent PDR inducers. Interestingly, protonophore PCP led to a persistent increase in the levels of a major ABC-transporter Pdr5p, while azole clotrimazole induced only a temporary increase. Together, our data provides an insight into the effects of the protonophores in the eukaryotes at the cellular level and support the idea that cells with activated PDR can be selected out upon conditions of energy limitations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10863-020-09849-1DOI Listing
October 2020

Mild depolarization of the inner mitochondrial membrane is a crucial component of an anti-aging program.

Proc Natl Acad Sci U S A 2020 03 9;117(12):6491-6501. Epub 2020 Mar 9.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia;

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1916414117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104298PMC
March 2020

Mitochondrial dynamics in yeast with repressed adenine nucleotide translocator AAC2.

Eur J Cell Biol 2020 Apr 4;99(2-3):151071. Epub 2020 Feb 4.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskiye Gory 1-40, Moscow, 119991, Russia; Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow, 119991, Russia. Electronic address:

The mitochondrial network structure dynamically adapts to cellular metabolic challenges. Mitochondrial depolarisation, particularly, induces fragmentation of the network. This fragmentation may be a result of either a direct regulation of the mitochondrial fusion machinery by transmembrane potential or an indirect effect of metabolic remodelling. Activities of ATP synthase and adenine nucleotide translocator (ANT) link the mitochondrial transmembrane potential with the cytosolic NTP/NDP ratio. Given that mitochondrial fusion requires cytosolic GTP, a decrease in the NTP/NDP ratio might also account for protonophore-induced mitochondrial fragmentation. For evaluating the contributions of direct and indirect mechanisms to mitochondrial remodelling, we assessed the morphology of the mitochondrial network in yeast cells with inhibited ANT. We showed that the repression of AAC2 (PET9), a major ANT gene in yeast, increases mitochondrial transmembrane potential. However, the mitochondrial network in this strain was fragmented. Meanwhile, AAC2 repression did not prevent mitochondrial fusion in zygotes; nor did it inhibit mitochondrial hyperfusion induced by Dnm1p inhibitor mdivi-1. These results suggest that the inhibition of ANT, rather than preventing mitochondrial fusion, facilitates mitochondrial fission. The protonophores were not able to induce additional mitochondrial fragmentation in an AAC2-repressed strain and in yeast cells with inhibited ATP synthase. Importantly, treatment with the ATP synthase inhibitor oligomycin A also induced mitochondrial fragmentation and hyperpolarization. Taken together, our data suggest that ATP/ADP translocation plays a crucial role in shaping of the mitochondrial network and exemplify that an increase in mitochondrial membrane potential does not necessarily oppose mitochondrial fragmentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2020.151071DOI Listing
April 2020

Genes Contribute to Environmental Stress Tolerance but Sensibilize Yeast Cells to Azoles.

Front Microbiol 2020 28;11:38. Epub 2020 Jan 28.

Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

Lam proteins transport sterols between the membranes of different cellular compartments. In the gene family consists of three pairs of paralogs. Because the function of paralogous genes can be redundant, the phenotypes of only a small number of gene deletions have been reported; thus, the role of these genes in yeast physiology is still unclear. Here, we surveyed the phenotypes of double and quadruple deletants of paralogous and genes that encode proteins localized in the junctions of the plasma membrane and endoplasmic reticulum. The quadruple deletant showed increased sterol content and a strong decrease in ethanol, heat shock and high osmolarity resistance. Surprisingly, the quadruple deletant and double deletion strain showed increased tolerance to the azole antifungals clotrimazole and miconazole. This effect was not associated with an increased rate of ABC-transporter substrate efflux. Possibly, increased sterol pool in the deletion strains postpones the effect of azoles on cell growth. Alternatively, deletions might alleviate the toxic effect of sterols as Lam proteins can transport toxic sterol biosynthesis intermediates into membrane compartments that are sensitive to these compounds. Our findings reveal novel biological roles of genes in stress tolerance and suggest that mutations in these genes may confer upregulation of a mechanism that provides resistance to azole antifungals in pathogenic fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.00038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997477PMC
January 2020

Effects of Sterols on the Interaction of SDS, Benzalkonium Chloride, and A Novel Compound, Kor105, with Membranes.

Biomolecules 2019 10 18;9(10). Epub 2019 Oct 18.

Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 1-40 Leninskie Gory, Moscow 119991, Russia.

Sterols change the biophysical properties of lipid membranes. Here, we analyzed how sterols affect the activity of widely used antimicrobial membrane-active compounds, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAC). We also tested a novel benzalkonium-like substance, Kor105. Our data suggest that benzalkonium and Kor105 disturb the ordering of the membrane lipid packaging, and this disturbance is dampened by cholesterol. The disturbance induced by Kor105 is stronger than that induced by BAC because of the higher rigidity of the Kor105 molecule due to a shorter linker between the phenyl group and quaternary nitrogen. On the contrary, individual SDS molecules do not cause the disturbance. Thus, in the tested range of concentrations, SDS-membrane interaction is not influenced by cholesterol. To study how sterols influence the biological effects of these chemicals, we used yeast strains lacking Lam1-4 proteins. These proteins transport sterols from the plasma membrane into the endoplasmic reticulum. We found that the mutants are resistant to BAC and Kor105 but hypersensitive to SDS. Together, our findings show that sterols influence the interaction of SDS versus benzalkonium chloride and Kor105 with the membranes in a completely different manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom9100627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843611PMC
October 2019

Replicative aging as a source of cell heterogeneity in budding yeast.

Mech Ageing Dev 2018 12 4;176:24-31. Epub 2018 Oct 4.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskiye Gory 1-40, Moscow 119991, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskiye Gory 1-73, Moscow 119991, Russia; Institute of Mitoengineering MSU, Leninskiye Gory 1, Moscow 119991, Russia.

While deviations from the optimal phenotype are deleterious, increased variation can prevent population extinction under severe stresses. Cell division asymmetry is an important source of microbial phenotypic heterogeneity. A consecutive set of asymmetric divisions can cause the gradual accumulation of deleterious factors and, at late stages, the death of old pole (mother) cells. This phenomenon is known as replicative aging. As the old cells are constantly being diluted by the progeny, the majority of a microbial population is represented by replicatively young cells. Therefore, early-age changes in yeast mother cells have a much greater impact on the integral performance of the microbial population than does functional deterioration at later ages. Here, we review the early manifestations of replicative aging in Saccharomyces cerevisiae mother cells that occur during the first ten cell cycles. Early age-dependent changes occur in stress resistance, genomic instability, protein aggregate levels, redox balance and metabolism. We speculate that some of these manifestations can be beneficial during stress exposure; therefore, early aging may be a bet-hedging mechanism. Together, the data suggest that the age component of variation in populations of asymmetrically dividing microorganisms is substantial and may play an important role in adaptations to changing environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mad.2018.09.001DOI Listing
December 2018

Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Science 2018 06;360(6395)

Institute of Microbiology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic.

Shlezinger (Reports, 8 September 2017, p. 1037) report that the common fungus , a cause of aspergillosis, undergoes caspase-dependent apoptosis-like cell death triggered by lung neutrophils. However, the technologies they used do not provide reliable evidence that fungal cells die via a protease signaling cascade thwarted by a fungal caspase inhibitor homologous to human survivin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aar6910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045345PMC
June 2018

Penetrating cations induce pleiotropic drug resistance in yeast.

Sci Rep 2018 05 25;8(1):8131. Epub 2018 May 25.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye Gory 1-40, Moscow, 119991, Russia.

Substrates of pleiotropic drug resistance (PDR) transporters can induce the expression of corresponding transporter genes by binding to their transcription factors. Penetrating cations are substrates of PDR transporters and theoretically may also activate the expression of transporter genes. However, the accumulation of penetrating cations inside mitochondria may prevent the sensing of these molecules. Thus, whether penetrating cations induce PDR is unclear. Using Saccharomyces cerevisiae as a model, we studied the effects of penetrating cations on the activation of PDR. We found that the lipophilic cation dodecyltriphenylphosphonium (CTPP) induced the expression of the plasma membrane PDR transporter genes PDR5, SNQ2 and YOR1. Moreover, a 1-hour incubation with CTPP increased the concentration of Pdr5p and Snq2p and prevented the accumulation of the PDR transporter substrate Nile red. The transcription factor PDR1 was required to mediate these effects, while PDR3 was dispensable. The deletion of the YAP1 or RTG2 genes encoding components of the mitochondria-to-nucleus signalling pathway did not prevent the CTPP-induced increase in Pdr5-GFP. Taken together, our data suggest (i) that the sequestration of lipophilic cations inside mitochondria does not significantly inhibit sensing by PDR activators and (ii) that the activation mechanisms do not require mitochondria as a signalling module.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-26435-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970188PMC
May 2018

Guidelines and recommendations on yeast cell death nomenclature.

Microb Cell 2018 Jan 1;5(1):4-31. Epub 2018 Jan 1.

Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15698/mic2018.01.607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772036PMC
January 2018

The contribution of Saccharomyces cerevisiae replicative age to the variations in the levels of Trx2p, Pdr5p, Can1p and Idh isoforms.

Sci Rep 2017 10 16;7(1):13220. Epub 2017 Oct 16.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye Gory 1-40, Moscow, 119991, Russia.

Asymmetrical division can be a reason for microbial populations heterogeneity. In particular, budding yeast daughter cells are more vulnerable to stresses than the mothers. It was suggested that yeast mother cells could also differ from each other depending on their replicative age. To test this, we measured the levels of Idh1-GFP, Idh2-GFP, Trx2-GFP, Pdr5-GFP and Can1-GFP proteins in cells of the few first, most represented, age cohorts. Pdr5p and Can1p were selected because of the pronounced mother-bud asymmetry for these proteins distributions, Trx2p as indicator of oxidative stress. Isocitrate dehydrogenase subunits Idh1p and Idh2p were assessed because their levels are regulated by mitochondria. We found a small negative correlation between yeast replicative age and Idh1-GFP or Idh2-GFP but not Trx2-GFP levels. Mitochondrial network fragmentation was also confirmed as an early event of replicative aging. No significant difference in the membrane proteins levels Pdr5p and Can1p was found. Moreover, the elder mother cells showed lower coefficient of variation for Pdr5p levels compared to the younger ones and the daughters. Our data suggest that the levels of stress-response proteins Pdr5p and Trx2p in the mother cells are stable during the first few cell cycles regardless of their mother-bud asymmetry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-13576-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643315PMC
October 2017

How do yeast sense mitochondrial dysfunction?

Microb Cell 2016 Sep 22;3(11):532-539. Epub 2016 Sep 22.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye Gory 1-40, Moscow 119991, Russia. ; Institute of Mitoengineering, Moscow State University, Leninskiye Gory 1, Moscow 119991, Russia.

Apart from energy transformation, mitochondria play important signaling roles. In yeast, mitochondrial signaling relies on several molecular cascades. However, it is not clear how a cell detects a particular mitochondrial malfunction. The problem is that there are many possible manifestations of mitochondrial dysfunction. For example, exposure to the specific antibiotics can either decrease (inhibitors of respiratory chain) or increase (inhibitors of ATP-synthase) mitochondrial transmembrane potential. Moreover, even in the absence of the dysfunctions, a cell needs feedback from mitochondria to coordinate mitochondrial biogenesis and/or removal by mitophagy during the division cycle. To cope with the complexity, only a limited set of compounds is monitored by yeast cells to estimate mitochondrial functionality. The known examples of such compounds are ATP, reactive oxygen species, intermediates of amino acids synthesis, short peptides, Fe-S clusters and heme, and also the precursor proteins which fail to be imported by mitochondria. On one hand, the levels of these molecules depend not only on mitochondria. On the other hand, these substances are recognized by the cytosolic sensors which transmit the signals to the nucleus leading to general, as opposed to mitochondria-specific, transcriptional response. Therefore, we argue that both ways of mitochondria-to-nucleus communication in yeast are mostly (if not completely) unspecific, are mediated by the cytosolic signaling machinery and strongly depend on cellular metabolic state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15698/mic2016.11.537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349209PMC
September 2016

Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.

J Cell Sci 2017 04 13;130(7):1274-1284. Epub 2017 Feb 13.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye Gory 1-40, Moscow 119991, Russia

Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes , or , implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.197269DOI Listing
April 2017

Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae.

Appl Environ Microbiol 2017 02 17;83(3). Epub 2017 Jan 17.

A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia

There are two superoxide dismutases in the yeast Saccharomyces cerevisiae-cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2 Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance.

Importance: Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.02759-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5244310PMC
February 2017

Alkylrhodamines enhance the toxicity of clotrimazole and benzalkonium chloride by interfering with yeast pleiotropic ABC-transporters.

FEMS Yeast Res 2016 06 4;16(4). Epub 2016 Apr 4.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskiye Gory 1-40, Moscow 119991, Russia Institute of Mitoengineering, Moscow State University, Leninskiye Gory 1, Moscow 119991, Russia.

ABC-transporters with broad substrate specificity are responsible for pathogenic yeast resistance to antifungal compounds. Here we asked whether highly hydrophobic chemicals with delocalized positive charge can be used to overcome the resistance. Such molecules efficiently penetrate the plasma membrane and accumulate inside the cells. We reasoned that these properties can convert an active efflux of the compounds into a futile cycle thus interfering with the extrusion of the antibiotics. To test this, we studied the effects of several alkylated rhodamines on the drug resistance of yeast Saccharomyces cerevisiae We found that octylrhodamine synergetically increases toxicity of Pdr5p substrate-clotrimazole, while the others were less effective. Next, we compared the contributions of three major pleiotropic ABC-transporters (Pdr5p, Yor1p, Snq2p) on the accumulation of the alkylated rhodamines. While all of the tested compounds were extruded by Pdr5p, Yor1p and Snq2p showed narrower substrate specificity. Interestingly, among the tested alkylated rhodamines, inactivation of Pdr5p had the strongest effect on the accumulation of octylrhodamine inside the cells, which is consistent with the fact that clotrimazole is a substrate of Pdr5p. As alkylated rhodamines were shown to be non-toxic on mice, our study makes them potential components of pharmacological antifungal compositions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/femsyr/fow030DOI Listing
June 2016

The double face of mitochondrial dysfunction.

Aging (Albany NY) 2016 Mar;8(3):420

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833134PMC
http://dx.doi.org/10.18632/aging.100923DOI Listing
March 2016

Mitochondrial retrograde signaling inhibits the survival during prolong S/G2 arrest in Saccharomyces cerevisiae.

Oncotarget 2015 Dec;6(42):44084-94

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.

Cell senescence is dependent on the arrest in cell cycle. Here we studied the role of mitochondrial retrograde response signaling in yeast cell survival under a prolonged arrest. We have found that, unlike G1, long-term arrest in mitosis or S phase results in a loss of colony-forming abilities. Consistent with previous observations, loss of mitochondrial DNA significantly increased the survival of arrested cells. We found that this was because the loss increases the duration of G1 phase. Unexpectedly, retrograde signaling, which is typically triggered by a variety of mitochondrial dysfunctions, was found to be a negative regulator of the survival after the release from S-phase arrest induced by the telomere replication defect. Deletion of retrograde response genes decreased the arrest-induced death in such cells, whereas deletion of negative regulator of retrograde signaling MKS1 had the opposite effect. We provide evidence that these effects are due to alleviation of the strength of the S-phase arrest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.6406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792543PMC
December 2015

Amiodarone induces cell wall channel formation in yeast Hansenula polymorpha.

Springerplus 2015 26;4:453. Epub 2015 Aug 26.

Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 1-40 Leninskie Gory, Moscow, 119992 Russia.

The yeast cell wall is constantly remodeled to enable cell growth and division. In this study, we describe a novel type of cell wall modification. We report that the drug amiodarone induces rapid channel formation within the cell wall of the yeast Hansenula polymorpha. Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface. Electron microphotographs show that amiodarone induces the formation of channels 50-80 nm in diameter in the cell wall that appear to be filled with intracellular material. Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels. We speculate that the observed channel formation acts to enable the secretion of mitochondrial material from the cell under stressful conditions, thus enabling adaptive changes to the extracellular environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40064-015-1185-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549368PMC
August 2015

Aging as an evolvability-increasing program which can be switched off by organism to mobilize additional resources for survival.

Curr Aging Sci 2015 ;8(1):95-109

Lomonosov Moscow State University, A. N. Belozersky Institute of Physico-Chemical Biology, Vorobyevy Gory 1, 119992 Moscow, Russia.

During the last decade, several pieces of convincing evidence were published indicating that aging of living organisms is programmed, being a particular case of programmed death of organism (phenoptosis). Among them, the following observations can be mentioned. (1) Species were described that show negligible aging. In mammals, the naked mole rat is the most impressive example. This is a rodent of mouse size living at least 10-fold longer than a mouse and having fecundity higher than a mouse and no agerelated diseases. (2) In some species with high aging rate, genes responsible for active organization of aging by poisoning of the organism with endogenous metabolites have been identified. (3) In women, standard deviations divided by the mean are the same for age of menarche (an event controlled by the ontogenetic program) and for age of menopause (an aging-related event). (4) Inhibitors of programmed cell death (apoptosis and necrosis) retard and in certain cases even reverse the development of age-dependent pathologies. (5) In aging species, the rate of aging is regulated by the individual which responds by changes in this rate to changes in the environmental conditions. In this review, we consider point (5) in detail. Data are summarized suggesting that inhibition of aging rate by moderate food restriction can be explained assuming that such restriction is perceived by the organism as a signal of future starvation. In response to this dramatic signal, the organism switches off such an optional program as aging, mobilizing in such a way additional reserves for survival. A similar explanation is postulated for geroprotective effects of heavy muscle work, a lowering or a rise in the external temperature, small amounts of metabolic poisons (hormesis), low doses of radiation, and other deleterious events. On the contrary, sometimes certain positive signals can prolong life by inhibiting the aging program in individuals who are useful for the community (e.g., geroprotective psychological factors). Similarly, dangerous individuals can be eliminated by programmed death due to operation of progeric psychological factors. The interplay of all these signals results in the final decision of the organism concerning its aging - to accelerate or to decelerate this process. Thus, paradoxically, such an originally counterproductive program as aging appears to be useful for the individual since this program can be switched off by the individual for a certain period of time, an action that thereby increases its resources in crucial periods of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
July 2016

Aging As An Evolvability-Increasing Program Which Can Be Switched Off By Organism To Mobilize Additional Resources For Survival.

Curr Aging Sci 2015 Apr 22. Epub 2015 Apr 22.

Lomonosov Moscow State University, A. N. Belozersky Institute of Physico-Chemical Biology, Vorobyevy Gory 1, 119992 Moscow, Russia.

During the last decade, several pieces of convincing evidence were published indicating that aging of living organisms is programmed, being a particular case of programmed death of organism (phenoptosis). Among them, the following observations can be mentioned [1]. Species were described that show negligible aging. In mammals, the naked mole rat is the most impressive example. This is a rodent of mouse size living at least 10-fold longer than a mouse and having fecundity higher than a mouse and no age-related diseases [2]. In some species with high aging rate, genes responsible for active organization of aging by poisoning of the organism with endogenous metabolites have been identified [3]. In women, standard deviations divided by the mean are the same for age of menarche (an event controlled by the ontogenetic program) and for age of menopause (an aging-related event) [4]. Inhibitors of programmed cell death (apoptosis and necrosis) retard and in certain cases even reverse the development of age-dependent pathologies [5]. In aging species, the rate of aging is regulated by the individual which responds by changes in this rate to changes in the environmental conditions. In this review, we consider point [5] in detail. Data are summarized suggesting that inhibition of aging rate by moderate food restriction can be explained assuming that such restriction is perceived by the organism as a signal of future starvation. In response to this dramatic signal, the organism switches off such an optional program as aging, mobilizing in such a way additional reserves for survival. A similar explanation is postulated for geroprotective effects of heavy muscle work, a lowering or a rise in the external temperature, small amounts of metabolic poisons (hormesis), low doses of radiation, and other deleterious events. On the contrary, sometimes certain positive signals can prolong life by inhibiting the aging program in individuals who are useful for the community (e.g., geroprotective psychological factors). Similarly, dangerous individuals can be eliminated by programmed death due to operation of progeric psychological factors. The interplay of all these signals results in the final decision of the organism concerning its aging - to accelerate or to decelerate this process. Thus, paradoxically, such an originally counterproductive program as aging appears to be useful for the individual since this program can be switched off by the individual for a certain period of time, an action that thereby increases its resources in crucial periods of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
April 2015

Dodecyltriphenylphosphonium inhibits multiple drug resistance in the yeast Saccharomyces cerevisiae.

Biochem Biophys Res Commun 2014 Aug 11;450(4):1481-4. Epub 2014 Jul 11.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobyevy Gory 1, Moscow, Russia; Institute of Mitoengineering, Moscow State University, Vorobyevy Gory 1, Moscow, Russia.

Multiple drug resistance pumps are potential drug targets. Here we asked whether the lipophilic cation dodecyltriphenylphosphonium (C12TPP) can interfere with their functioning. First, we found that suppression of ABC transporter gene PDR5 increases the toxicity of C12TPP in yeast. Second, C12TPP appeared to prevent the efflux of rhodamine 6G - a fluorescent substrate of Pdr5p. Moreover, C12TPP increased the cytostatic effects of some other known Pdr5p substrates. The chemical nature of C12TPP suggests that after Pdr5p-driven extrusion the molecules return to the plasma membrane and then into the cytosol, thus effectively competing with other substrates of the pump.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2014.07.017DOI Listing
August 2014

Early manifestations of replicative aging in the yeast .

Microb Cell 2014 Jan 6;1(1):37-42. Epub 2014 Jan 6.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobyevy Gory 1, Moscow, Russia. ; Institute of Mitoengineering, Moscow State University, Vorobyevy Gory 1, Moscow, Russia.

The yeast is successfully used as a model organism to find genes responsible for lifespan control of higher organisms. As functional decline of higher eukaryotes can start as early as one quarter of the average lifespan, we asked whether can be used to model this manifestation of aging. While the average replicative lifespan of mother cells ranges between 15 and 30 division cycles, we found that resistances to certain stresses start to decrease much earlier. Looking into the mechanism, we found that knockouts of genes responsible for mitochondria-to-nucleus (retrograde) signaling, or significantly decrease the resistance of cells that generated more than four daughters, but not of the younger ones. We also found that even young mother cells frequently contain mitochondria with heterogeneous transmembrane potential and that the percentage of such cells correlates with replicative age. Together, these facts suggest that retrograde signaling starts to malfunction in relatively young cells, leading to accumulation of heterogeneous mitochondria within one cell. The latter may further contribute to a decline in stress resistances.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15698/mic2014.01.122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349164PMC
January 2014

Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations.

Biochim Biophys Acta 2014 Jan 27;1837(1):149-58. Epub 2013 Sep 27.

A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Moscow 119991, Russia; Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Moscow 119991, Russia.

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbabio.2013.09.011DOI Listing
January 2014

Roles of mitochondrial dynamics under stressful and normal conditions in yeast cells.

Oxid Med Cell Longev 2013 14;2013:139491. Epub 2013 Jul 14.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobyevy Gory 1, Moscow 119992, Russia.

Eukaryotic cells contain dynamic mitochondrial filaments: they fuse and divide. Here we summarize data on the protein machinery driving mitochondrial dynamics in yeast and also discuss the factors that affect the fusion-fission balance. Fission is a general stress response of cells, and in the case of yeast this response appears to be prosurvival. At the same time, even under normal conditions yeast mitochondria undergo continuous cycles of fusion and fission. This seems to be a futile cycle and also expensive from the energy point of view. Why does it exist? Benefits might be the same as in the case of sexual reproduction. Indeed, mixing and separating of mitochondrial content allows mitochondrial DNA to segregate and recombine randomly, leading to high variation in the numbers of mutations per individual mitochondrion. This opens a possibility for effective purifying selection-elimination of mitochondria highly contaminated by deleterious mutations. The beneficial action presumes a mechanism for removal of defective mitochondria. We argue that selective mitochondrial autophagy or asymmetrical distribution of mitochondria during cell division could be at the core of such mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2013/139491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728513PMC
February 2014

Structure-based design of small-molecule ligands of phosphofructokinase-2 activating or inhibiting glycolysis.

ChemMedChem 2013 Aug 28;8(8):1322-9. Epub 2013 Jun 28.

Quantum Pharmaceuticals LLC, Kosmonavta Volkova 6A, 1205, Moscow 125171, Russia.

Glycolysis lies at the basis of metabolism and cell energy supply. The disregulation of glycolysis is involved in such pathological processes as cancer proliferation, neurodegenerative diseases, and amplification of ischemic damage. Phosphofructokinase-2 (PFK-2), a bifunctional enzyme and regulator of glycolytic flux, has recently emerged as a promising anticancer target. Herein, the computer-aided design of a new class of aminofurazan-triazole regulators of PFK-2 is described along with the results of their in vitro evaluation. The aminofurazan-triazoles differ from other recently described inhibitors of PFK-2 and demonstrate the ability to modulate glycolytic flux in rat muscle lysate, producing a twofold decrease by inhibitors and fourfold increase by activators. The most potent compounds in the series were shown to inhibit the kinase activity of the hypoxia-inducible form of PFK-2, PFKFB3, as well as proliferation of HeLa, lung adenocarcinoma, colon adenocarcinoma, and breast cancer cells at concentrations in the low micromolar range.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201300154DOI Listing
August 2013

In search of novel highly active mitochondria-targeted antioxidants: thymoquinone and its cationic derivatives.

FEBS Lett 2013 Jun 10;587(13):2018-24. Epub 2013 May 10.

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, Institute of Mitoengineering, Vorobyevy Gory 1, Moscow 119992, Russia.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2-demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria-targeted antioxidants effective in treating a large number of age-related pathologies. This review summarizes new data on the antioxidant and some other properties of membrane-penetrating cationic compounds where 2-demethylplastoquinone substitutes for plastoquinone. It was found that such a substitution significantly increases a window between anti- and prooxidant concentrations of the conjugates. Like the original plastoquinone derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate through model and natural membranes, specifically accumulate in mitochondria in an electrophoretic fashion, and strongly inhibit H2O2-induced apoptosis at pico- and nanomolar concentrations in cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promising mitochondria-targeted drugs of the quinone series.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2013.04.043DOI Listing
June 2013

Penetrating cations enhance uncoupling activity of anionic protonophores in mitochondria.

PLoS One 2013 23;8(4):e61902. Epub 2013 Apr 23.

M.V. Lomonosov Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H(+) ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C12TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C12TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C12TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061902PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633956PMC
November 2013

Mitochondrially-encoded protein Var1 promotes loss of respiratory function in Saccharomyces cerevisiae under stressful conditions.

Eur J Cell Biol 2013 Apr-May;92(4-5):169-74. Epub 2013 Mar 13.

Faculty of Bioengineering and Bioinformatics, Moscow State University, Vorobyevy Gory 1, Moscow, Russia.

Stressed Saccharomyces cerevisiae cells easily lose respiratory function due to deletions in mitochondrial DNA, and this increases their general stress resistance. Is the loss active? We found that erythromycin (an inhibitor of mitochondrial translation) prevents the loss in control cells but not in the ones expressing mitochondrially-encoded protein Var1 in the nucleus. Var1 is a component of mitochondrial ribosomes; it is hydrophilic, positively charged, and prone to aggregation. Addition of DNase altered Var1 content in a preparation of mitochondrial nucleoids. Our data indicate that Var1 physically interacts with mitochondrial DNA and under stress negatively regulates its maintenance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2013.02.001DOI Listing
October 2013

Mitochondrial signaling in Saccharomyces cerevisiae pseudohyphae formation induced by butanol.

FEMS Yeast Res 2013 Jun 19;13(4):367-74. Epub 2013 Mar 19.

Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia.

Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells--pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP-synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol-induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria-activated signaling pathways appear to converge at Mih1p level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1567-1364.12039DOI Listing
June 2013

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers.

J Biol Chem 2011 May 30;286(20):17831-40. Epub 2011 Mar 30.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory 1, Moscow 119991, Russia.

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.212837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093858PMC
May 2011