Karin Nowikovsky

Standard animal behavior paradigms incompletely mimic nature and thus limit our understanding of behavior and brain function. Virtual reality (VR) can help, but it poses challenges. Typical VR systems require movement restrictions but disrupt sensorimotor experience, causing neuronal and behavioral alterations. We report the development of FreemoVR, a VR system for freely moving animals. We validate immersive VR for mice, flies, and zebrafish. FreemoVR allows instant, disruption-free environmental reconfigurations and interactions between real organisms and computer-controlled agents. Using the FreemoVR platform, we established a height-aversion assay in mice and studied visuomotor effects in Drosophila and zebrafish. Furthermore, by photorealistically mimicking zebrafish we discovered that effective social influence depends on a prospective leader balancing its internally preferred directional choice with social interaction. FreemoVR technology facilitates detailed investigations into neural function and behavior through the precise manipulation of sensorimotor feedback loops in unrestrained animals.

Loss of the MDM38 gene product in yeast mitochondria results in a variety of phenotypic effects including reduced content of respiratory chain complexes, altered mitochondrial morphology and loss of mitochondrial K(+)/H(+) exchange activity resulting in osmotic swelling. By use of doxycycline-regulated shut-off of MDM38 gene expression, we show here that loss of K(+)/H(+) exchange activity and mitochondrial swelling are early events, associated with a reduction in membrane potential and fragmentation of the mitochondrial reticulum. Changes in the pattern of mitochondrially encoded proteins are likely to be secondary to the loss of K(+)/H(+) exchange activity. The use of a novel fluorescent biosensor directed to the mitochondrial matrix revealed that the loss of K(+)/H(+) exchange activity was immediately followed by morphological changes of mitochondria and vacuoles, the close association of these organelles and finally uptake of mitochondrial material by vacuoles. Nigericin, a K(+)/H(+) ionophore, fully prevented these effects of Mdm38p depletion. We conclude that osmotic swelling of mitochondria triggers selective mitochondrial autophagy or mitophagy.

Cardiac and skeletal muscle critically depend on mitochondrial energy metabolism for their normal function. Recently, we showed that apoptosis-inducing factor (AIF), a mitochondrial protein implicated in programmed cell death, plays a role in mitochondrial respiration. However, the in vivo consequences of AIF-regulated mitochondrial respiration resulting from a loss-of-function mutation in Aif are not known. Here, we report tissue-specific deletion of Aif in the mouse. Mice in which Aif has been inactivated specifically in cardiac and skeletal muscle exhibit impaired activity and protein expression of respiratory chain complex I. Mutant animals develop severe dilated cardiomyopathy, heart failure, and skeletal muscle atrophy accompanied by lactic acidemia consistent with defects in the mitochondrial respiratory chain. Isolated hearts from mutant animals exhibit poor contractile performance in response to a respiratory chain-dependent energy substrate, but not in response to glucose, supporting the notion that impaired heart function in mutant animals results from defective mitochondrial energy metabolism. These data provide genetic proof that the previously defined cell death promoter AIF has a second essential function in mitochondrial respiration and aerobic energy metabolism required for normal heart function and skeletal muscle homeostasis.

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Δ mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Δ mitochondria have increased cation contents, particularly K+ and low-membrane-potential Δψ. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K+/H+ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K+ homeostasis and organelle volume control. The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Δ mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Δ mitochondria have increased cation contents, particularly K+ and low-membrane-potential Δψ. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K+/H+ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K+ homeostasis and organelle volume control. Respiring mitochondria maintain a membrane potential (Δψ) 1The abbreviations used are: Δψ, membrane potential; KOAc, potassiumacetate; HA, hemagglutin; GFP, green fluorescent protein; YPGal, yeast extract, bacto peptone, 2% galactose; YPD, yeast extract, bacto peptone, 2% dextrose; SMP, submitochondrial particles; DCCD, dicyclohexylcarbodiimide; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; TES, N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid; A23187, 4-bromo-calcium ionophore A23187; TM, transmembrane.1The abbreviations used are: Δψ, membrane potential; KOAc, potassiumacetate; HA, hemagglutin; GFP, green fluorescent protein; YPGal, yeast extract, bacto peptone, 2% galactose; YPD, yeast extract, bacto peptone, 2% dextrose; SMP, submitochondrial particles; DCCD, dicyclohexylcarbodiimide; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; TES, N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid; A23187, 4-bromo-calcium ionophore A23187; TM, transmembrane. of –150 to –180 mV (Δψ, inside negative). This high Δψ constitutes a large driving force for the electrophoretic influx of cations, either through specific channels or by diffusion through the membrane. Several cation channels have been characterized physiologically (reviewed in Refs. 1Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1335) Google Scholar and 2Garlid K.D. Paucek P. Biochim. Biophys. Acta. 2003; 1606: 23-41Crossref PubMed Scopus (305) Google Scholar), and recently, a single one has been identified molecularly (3Kolisek M. Zsurka G. Samaj J. Weghuber J. Schweyen R.J. Schweigel M. EMBO J. 2003; 22: 1235-1244Crossref PubMed Scopus (170) Google Scholar). These transport systems seem to have intrinsic control mechanisms which ensure that the matrix cation concentrations stay within physiological ranges, far below chemical equilibrium. Diffusive permeability of the inner mitochondrial membrane to ions is generally low but physiologically significant, as it lowers the pH gradient and membrane potential. Moreover, if not counteracted by extrusion, steadily increasing concentrations of matrix cations (and of compensating anions) will lead to an imbalance of osmotic pressure across the inner mitochondrial membrane. As a consequence, water will pass through the membrane, causing excessive swelling and eventual rupture of the organelle (1Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1335) Google Scholar, 2Garlid K.D. Paucek P. Biochim. Biophys. Acta. 2003; 1606: 23-41Crossref PubMed Scopus (305) Google Scholar, 4Brierley G.P. Jung D.W. J. Bioenerg. Biomembr. 1988; 20: 193-209Crossref PubMed Scopus (29) Google Scholar). As first proposed by P. Mitchell (5Mitchell P. Naturwissenschaften. 1961; 191: 144-148Google Scholar), mitochondria have carrier systems allowing the electroneutral exchange of cations against H+ (and anions against OH–). These exchangers counteract the Δψ-driven cation leakage of the membrane and also cation imbalances due to changes in mitochondrial physiology. Mitochondrial cation distribution is, therefore, a steady state, in which the accumulation ratio is modulated by the relative rates of cation influx and efflux by means of separate pathways. Many physiological studies have been devoted to cation/H+ exchange systems (reviewed in Ref. 1Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1335) Google Scholar). With respect to the most abundant cations in cells and mitochondria, K+ (150 mm) and Na+ (5 mm), researchers agree on the existence of two separate antiporters in mammalian cells, a selective Na+/H+ exchanger, and an unselective K+/H+ exchanger transporting virtually all alkali ions. Given the particularly high concentration of K+ in cells and mitochondria, the unselective exchanger is referred to most commonly as the K+/H+ exchanger (reviewed in Ref. 1Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1335) Google Scholar). This exchanger has pronounced sensitivity to matrix [Mg2+](Ki of 0.3–0.4 mm in mammalian mitochondria), timolol, and quinine. Proteins of apparent molecular masses of 82 and 59 kDa constitute the unselective mitochondrial K+/H+ exchanger and the selective Na+/H+ exchanger, respectively (6Li X.Q. Hegazy M.G. Mahdi F. Jezek P. Lane R.D. Garlid K.D. J. Biol. Chem. 1990; 265: 15316-15322Abstract Full Text PDF PubMed Google Scholar, 7Jezek P. Mahdi F. Garlid K.D. J. Biol. Chem. 1990; 265: 10522-10526Abstract Full Text PDF PubMed Google Scholar). Attempts to identify the gene encoding the K+/H+ have not been successful yet (2Garlid K.D. Paucek P. Biochim. Biophys. Acta. 2003; 1606: 23-41Crossref PubMed Scopus (305) Google Scholar), and a report on the identification of the yeast NHE2 and its mammalian homolog NHE6 as encoding the mitochondrial Na+/H+ exchanger (8Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) have been questioned recently (9Brett C.L. Wei Y. Donowitz M. Rao R. Am. J. Physiol. 2002; 282: C1031-C1041Crossref PubMed Scopus (141) Google Scholar). In the course of characterizing a set of yeast genes potentially encoding mitochondrial cation transport proteins (10Waldherr M. Ragnini A. Jank B. Teply R. Wiesenberger G. Schweyen R.J. Curr. Genet. 1993; 24: 301-306Crossref PubMed Scopus (60) Google Scholar) we focused on the yeast genes MRS7 and YOL027, as well as their human homolog LETM1 (leucine zipper/EF-hand-containing trans-membrane domain; Ref. 11Endele S. Fuhry M. Pak S.J. Zabel B.U. Winterpacht A. Genomics. 1999; 60: 218-225Crossref PubMed Scopus (122) Google Scholar), which are representatives of a novel eukaryotic gene family with hitherto unknown function. Hemizygous deletion of a region on human chromosome 4 (4p16.3), including LETM1 and several other genes, causes the Wolf-Hirschhorn syndrome. Recent data reveal that the full Wolf-Hirschhorn syndrome phenotype, including neuromuscular features, such as seizures correlates with the deletion of the LETM1 gene (12Zollino M. Lecce R. Fischetto R. Murdolo M. Faravelli F. Selicorni A. Butte C. Memo L. Capovilla G. Neri G. Am. J. Hum. Genet. 2003; 72: 590-597Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). We report here on the mitochondrial localization of the human LetM1 protein and of its yeast homologs, Yol027p and Ypr125p (Mrs7p), on their functional homology, and on the effects resulting from the disruption of the yeast genes on mitochondrial functions. The results indicate a role of Yol027p in mitochondrial K+ homeostasis. As compared with wild-type mitochondria, mutant yol027Δ mitochondria exhibit severely reduced potassium acetate (KOAc)-induced swelling, which is indicative of a lack or reduction in K+/H+-exchange activity. We discuss the possibility that YOL027 encodes either the K+/H+ exchanger itself or an essential cofactor thereof. Strains, Plasmids, and Media—The following strains of Saccharomyces cerevisiae were used: GA74–1A (13Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), DBY747 (ATCC no. 204659), and W303 (ATCC no. 201239), all of which served as wild-type strains, and DBY747 mrs2Δ (14Wiesenberger G. Waldherr M. Schweyen R.J. J. Biol. Chem. 1992; 267: 6963-6969Abstract Full Text PDF PubMed Google Scholar). Yeast growth media were as described previously (14Wiesenberger G. Waldherr M. Schweyen R.J. J. Biol. Chem. 1992; 267: 6963-6969Abstract Full Text PDF PubMed Google Scholar). Hemagglutin (HA)-tagged and Green Fluorescent Protein (GFP)-tagged Genes—A transposon-tagged YOL027c-containing DNA fragment (15Ross-Macdonald P. Coelho P.S. Roemer T. Agarwal S. Kumar A. Jansen R. Cheung K.H. Sheehan A. Symoniatis D. Umansky L. Heidtman M. Nelson F.K. Iwasaki H. Hager K. Gerstein M. Miller P. Roeder G.S. Snyder M. Nature. 1999; 402: 413-418Crossref PubMed Scopus (454) Google Scholar) was used to replace the chromosomal copy of YOL027c in strain GA74–1A. Upon Cre-mediated recombination, a variant of the YOL027c gene was obtained which had a triple HA-tag inserted inframe after codon 469. This HA-tagged version of the YOL027c gene had no apparent phenotypic effect on the growth of the mutant strain compared with the isogenic wild-type strain. The YOL027c gene sequence (nucleotides –426 (relative to the start codon) to +1721) was PCR-amplified from W303 genomic DNA by use of an oligonucleotide 5′ primer carrying a natural SacI site and a 3′ primer that introduced a PstI site upstream of the stop codon. The SacI-PstI fragment of this product was inserted into YCp33-HA vector, resulting in plasmid YCp-YOL027-HA. YPR125 coding sequence (1356 nucleotides from 1 to +1356) was PCR-amplified from the same yeast strain, introducing recognition sites for EcoRI and SalI, followed by insertion of this fragment into the EcoRI and SalI sites of the centromeric vector pUG35. The resulting plasmid pUG35-YPR125-GFP expressed the YPR125 gene under the MET25 promoter and in-frame with the GFP coding sequence following at its 3′ end. The YEp-YPR125 construct (YEp-MW7) had been cloned by Waldherr et al. (10Waldherr M. Ragnini A. Jank B. Teply R. Wiesenberger G. Schweyen R.J. Curr. Genet. 1993; 24: 301-306Crossref PubMed Scopus (60) Google Scholar). To clone the human LETM1 cDNA, SuperScript II reverse transcriptase (Invitrogen) and an oligo(dT) primer were used for first-strand synthesis on poly(A)-enriched template RNA isolated from a PA-1 human teratocarcinoma cell line. PCR fragments covering the entire coding sequences (from nucleotide 1 to +2590) were amplified, and the purified PCR product was cloned to the pGEM-T vector (Promega), and a C-terminal HA-tag was added by PCR. The PCR product was digested with XhoI and HindIII and cloned into the corresponding sites of the yeast expression vector, pVT103-U. An XhoI-EcoRI PCR fragment was cloned into the pEGFP-N1 mammalian expression vector, thus creating an in-frame fusion with the enhanced GFP (EGFP) sequence carried on the vector. Gene Deletion—Complete disruptions of YOL027, MRS2, and MRS7 were performed according to the one-step replacement protocol described in Ref. 16Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2232) Google Scholar in the diploid yeast strain W303 and the haploid DBY747. Disruption of YOL027 resulted in a deletion of 1702 nucleotides (from the start codon to nucleotide –19, relative to the stop codon) of the YOL027 open reading frame (named yol027Δ mutant). Spores derived from this diploid strain were found to be viable. A disruption of the same size then was obtained in DBY747 (haploid). Disruption of the open reading frames MRS2 (mrs2Δ) and MRS7 (mrs7Δ) resulted in deletions of 1218 nucleotides (from nucleotide –49 relative to the start codon to nucleotide –243 relative to the stop codon) and of 1356 nucleotides (from the start to the stop codon). 2L. Zotova, unpublished data. W303 yol027Δmrs2Δ and yol027Δmrs7Δ double-mutant strains were then obtained by crossing the yol027Δ strain with the mrs2Δ or ypr125Δ strain, respectively. Diploids were sporulated, and the haploid double mutants were identified among the meiotic progeny by screening for the appropriate combination of disruption markers. Isolation and Subfractionation of Mitochondria—GA74–1A were grown in lactate medium (17Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar), W303 cells in complete YPGal medium or synthetic S-galactopyranoside (S-Gal) medium containing 2% galactose. Strain DBY747 was cultivated in YPD medium. For ion-influx measurements, cells were grown to stationary phase; for all other applications, cells were harvested at A600 of 1. Mitochondria were prepared according to Ref. 18Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (301) Google Scholar and suspended in breaking buffer (0.6 m sorbitol HEPES-KOH, pH 7.4) prior to further use. Na2CO3 extraction of proteins from membranes and mitoplast preparation was as described in Refs. 20Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1381) Google Scholar and 18Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (301) Google Scholar, respectively. Yeast mitochondria obtained by differential centrifugation were diluted to a protein concentration of 10 mg/ml with 10 mm HEPES-Tris-Cl, pH 7.4, and a final osmolarity of 0.1 m. After a 20-min incubation on ice, the resulting mitoplasts were collected by centrifugation (40,000 × g for 10 min at 4 °C). To obtain submitochondrial particles (SMP), mitoplasts were resuspended in sucrose buffer (250 mm sucrose, 10 mm Tris-Cl, pH 7.4). The mitoplast suspension was sonified for 3 min with maximum intensity in a Bandelin sonicator UW70/GM70. After removing unbroken mitochondria (10-min centrifugation at 10,000 × g), SMPs were collected by centrifugation at 100,000 × g for 1 h and resuspended in 1 ml of sucrose buffer. Antibodies used for immunodetection were as described in Ref. 19Jarosch E. Tuller G. Daum G. Waldherr M. Voskova A. Schweyen R.J. J. Biol. Chem. 1996; 271: 17219-17225Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar or kindly provided by Hans van der Spek (Tom44p), Jan Brix (Tom70p), Doron Rapaport (Aac2p), and Thomas D. Fox (Yme1p). Protein-anti-body complexes were visualized on Western blots using the SuperSignal™ West Pico system (Pierce). For fluorimetric determination of cation concentrations, measurements of matrix concentrations of free Mg2+ and Ca2+ ([Mg2+]m and [Ca2+]m) were performed in mitochondria from the DBY747 background as described in Ref. 3Kolisek M. Zsurka G. Samaj J. Weghuber J. Schweyen R.J. Schweigel M. EMBO J. 2003; 22: 1235-1244Crossref PubMed Scopus (170) Google Scholar. KOAc-induced Swelling of Isolated Mitochondria—Mitochondria were prepared from DBY747 or W303 wild-type cells and from isogenic yol027Δ mutant cells, resuspended in 0.6 m sorbitol buffer Tris-Cl, pH 7.4, to a final concentration of 10 mg of protein per ml. Aliquots of 100 μl of this suspension were incubated for 5 min at 25 °C with antimycin A (final concentration of 5 μm) and then transferred into cuvettes containing 1 ml of swelling buffer (55 mm KOAc, 5 mm TES, 0.1 mm EGTA, and 0.1 mm EDTA). Recording of A540 (Hitachi U-2000 spectrophotometer) was started immediately thereafter. To deplete mitochondria of endogenous Mg2+, the 4-bromo-calcium ionophore A23187 (0.5 μm) and EDTA (10 mm) were added prior to the KOAc treatment. To observe inhibition of swelling carbonyl cyanide m-chlorophenylhydrazone (CCCP), quinine, or dicyclohexylcarbodiimide (DCCD) (final concentrations of 0.5 μm, 200 μm, and 1 mm, respectively) were added to A23187/EDTA-treated mitochondria prior to the KOAc treatment. Confocal Fluorescence Microscopy—Cells transformed with plasmids expressing GFP fusion proteins were stained with 10 μm rhodamine B hexyl ester (Molecular Probes) or 25 nm Mito-Tracker red chloromethyl-X-rosamine and examined by laser confocal microscopy using a Leica TCS4D laser confocal microscope. GFP and rhodamine B were excited by 488 and 543 nm laser lines, respectively, and detected simultaneously at their emission maxima. Mitochondrial polarization was observed by laser confocal microscopy as described in Ref. 3Kolisek M. Zsurka G. Samaj J. Weghuber J. Schweyen R.J. Schweigel M. EMBO J. 2003; 22: 1235-1244Crossref PubMed Scopus (170) Google Scholar. Electron Microscopy—W303 cells were grown at 28 °C in YPGal to an A600 of 1.2, fixed for 30 min in 3.7% formaldehyde, spheroblasted with zymolyase at 0.5 mg/g of cells, and washed in phosphate-buffered saline. Spheroblasts were pelleted and resuspended in 2% glutaraldehyde in 0.15 m Sorensen′s buffer (pH 7.4) for postfixation overnight at 4 °C. Subsequently, the cell suspensions were filled into cellulose tubes (200 μm in diameter), infiltrated with 1% OsO4 for 1 h, dehydrated in ethanol, and embedded in epoxy resin Agar 100 (Agar Scientific Ltd, UK). Thin sections were cut on a Reichert Ultracut S microtome, mounted on copper grids, and contrasted by uranyl acetate and lead citrate. Grids were examined at 60 kV using a JEM-1210 electron microscope (Jeol Ltd., Japan). LetM1p, YOL027p, and Ypr125p Are Members of a Novel Eukaryotic Protein Family—LETM1, a human open reading frame of unknown function, is part of most deletions in chromosome 4 causing Wolf-Hirschhorn syndrome. It encodes a protein of 83.4 kDa (11Endele S. Fuhry M. Pak S.J. Zabel B.U. Winterpacht A. Genomics. 1999; 60: 218-225Crossref PubMed Scopus (122) Google Scholar). Homologs of this protein have been detected in all heavily sequenced eukaryotes. The genome of the yeast S. cerevisiae contains two open reading frames (YOL027 and YPR125) encoding LETM1 homologs of 573 and 454 amino acids, respectively, with about 40% sequence identity. Although the size variation among these homologs is high, lower eukaryotes, animals, and plants have at least one predicted transmembrane domain (Fig. 1A). In addition, most proteins in animals and plants have one or two predicted EF-hand calcium-binding domains in their C-terminal extensions, and the mammalian ones contain a leucine zipper region (Fig. 1A), as first noted in Ref. 11Endele S. Fuhry M. Pak S.J. Zabel B.U. Winterpacht A. Genomics. 1999; 60: 218-225Crossref PubMed Scopus (122) Google Scholar. Full-sequence alignments of homologs from plant, human, and yeast (Fig. 1B) reveal that members of this new protein family are highly conserved in their middle parts (about 40% amino acid identity). The region predicted to contain a transmembrane domain is particularly well conserved (TM, boxed in Fig. 1B). Three prolines within the putative α helical transmembrane sequence (Fig. 1C) are remarkable. Prolines, forming molecular hinges, have been observed repeatedly in transmembrane α helices of proteins, notably in ion channels and G protein-coupled receptors (21Sansom M.S. Weinstein H. Trends Pharmacol. Sci. 2000; 21: 445-451Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). LetM1p and Ypr125p Localize to Mitochondria—The human LETM1 gene and the yeast YPR125 gene were C-terminally tagged with the GFP-epitope. The LetM1-GFP fusion protein was transiently expressed from the vector pEGFP-N1 in the mouse NIH/3T3 embryonic fibroblast cell line. Fluorescence confocal microscopy revealed the co-localization of the GFP fluorescence with Mito-Tracker fluorescence of mitochondria (Fig. 2, a–c). When expressed under control of the methionine promoter from a yeast low-copy plasmid (pUG35), the Ypr125-GFP fusion protein co-localized with Mito-Tracker fluorescence, visualizing a distinct tubular network typical of yeast mitochondria (Fig. 2, d–f). Yol027p Is an Integral Protein of Mitochondrial Inner Membrane—A YOL027-HA allele (triple HA tag C-terminally fused to the YOL027 open reading frame and inserted at the chromosomal locus; see “Experimental Procedures”) was used to determine the subcellular localization of Yol027p by cell fractionation and immunoblotting. Total cell content (T), post-mitochondrial supernatant (C), and mitochondrial (M) fractions were separated by SDS-PAGE and analyzed by immunoblotting (Fig. 3A, lanes T, C, M). The cytosolic fraction was characterized by the presence of hexokinase Hxk1p, a soluble protein. Yol027-HAp was found exclusively in the total cell content and mitochondrial fractions, as were the ADP/ATP carrier Aac2p, an integral protein of the inner membrane, and the β subunit of the F1 ATPase, F1β, a protein associated with the matrix side of the inner membrane. Treatment of mitochondria by alkaline sodium carbonate (20Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1381) Google Scholar) solubilized the membrane-associated ATPase subunit F1β (Fig. 3A, lane SN), but not the integral membrane protein Aac2p (Fig. 3A, lane P). Yol027-HA protein also stayed in the pellet fraction, thus qualifying it as an integral membrane protein. Cell fractionation, sodium carbonate extraction, and immunoblotting also revealed that Ypr125-GFP and LetM1-GFP behaved as integral proteins of a mitochondrial membrane (data not shown). To further determine to which of the two mitochondrial membranes Yol027-HA localizes, whole mitochondria, mitoplasts, and SMPs of a yol027Δ mutant strain expressing a C-terminally HA-tagged Yol027 protein from a single-copy plasmid were obtained, and the accessibility of their proteins by proteinase K was studied (Fig. 3B). In whole mitochondria, all tested proteins were protease resistant, except Tom70p, an outer membrane protein protruding to the surface. Upon disintegration of the membranes by Triton X-100, all proteins were digested by proteinase K, showing that none of them was intrinsically protease-resistant. Mitoplasts were characterized by (i) the absence of Tom70p, pointing to an efficient removal of the outer membrane, (ii) by protection of the matrix-sided integral membrane protein Tim44 from degradation by proteinase K, (iii) by the degradation of Yme1p, an inner membrane protein with domains exposed to the intermembrane space and the matrix, and (iv) shortening of Aac2p, the ADP/ATP carrier, an inner mitochondrial membrane protein partially exposed to the outside of mitoplasts. Mitoplasts contained Yol027p, but in a proteinase K-resistant form, which implies that no part of this protein protrudes to the intermembrane space to such an extent that it is rendered protease-sensitive. Sonication of mitoplasts is known to result in the formation of SMPs with a majority of inside-out vesicles (22Godinot C. Gautheron D.C. Methods Enzymol. 1979; 55: 112-114Crossref PubMed Scopus (20) Google Scholar). Consistently, we found that Tim44p became protease-sensitive, whereas Aac2p lost its protease-sensitivity. The presence of Yol027-HA in these SMPs confirmed its nature as a membrane protein, and its protease-sensitivity indicated that it was exposed to the surface of the SMPs. This change in protease sensitivity of Yol027-HA and Aac2p indicates that sonication of mitoplasts under the conditions used here led to a very large fraction of inside-out particles, allowing the conclusion that the C terminus of Yol027p is located in the mitochondrial matrix. Disruption of the YOL027 Gene—To investigate the function of Yol027p, the YOL027 coding sequence was replaced by the HIS3MX6 cassette in the diploid yeast strain W303 (see “Experimental Procedures”). After sporulation of the resulting heterozygous strain and tetrad dissection, yol027Δ spores were found to exhibit reduced growth on non-fermentable carbon sources (YPEG) at 28 °C and nearly no growth at 37 °C (Fig. 4) and at 18 °C (data not shown). Fermentative growth of the mutant (on YPD) was also reduced, as compared with that of the isogenic wild-type (Fig. 4). When grown on glucose containing media, yol027Δ strains were mitotically unstable, throwing off rho– cells (having macro-deletions in mitochondrial DNA) at a moderate rate (data not shown). Disruption of YPR125 had no apparent phenotype. Disruption of both YOL027 and YPR125 (yol027Δ ypr125Δ mutant) led to a phenotype indistinguishable from the one exhibited by the yol027Δ mutant (data not shown). Because YOL027 and YPR125 are multicopy suppressors of the mrs2Δ petite phenotype, defective in mitochondrial Mg2+ influx (3Kolisek M. Zsurka G. Samaj J. Weghuber J. Schweyen R.J. Schweigel M. EMBO J. 2003; 22: 1235-1244Crossref PubMed Scopus (170) Google Scholar, 10Waldherr M. Ragnini A. Jank B. Teply R. Wiesenberger G. Schweyen R.J. Curr. Genet. 1993; 24: 301-306Crossref PubMed Scopus (60) Google Scholar), we also investigated the phenotypes of a yol027Δ mrs2Δ double mutant. This mutant was unable to grow on non-fermentable substrate at any temperature and proved to be rhoo (devoid of mitochondrial DNA; data not shown). Simultaneous deletion of YOL027 and MRS2 thus results in a more pronounced (synthetic) growth defect than single deletions of each of these two genes. Functional Homology of Yeast and Human LetM1p—To find out if the human LetM1p is a functional homolog of Yol027p, we transformed a yol027Δ strain with a plasmid expressing the LETM1 gene from the strong, constitutive ADH1 promoter on a multicopy plasmid ((LETM1)n). As a control, the strain was also transformed with the empty plasmid and a plasmid containing the YOL027 coding region. As shown in Fig. 4, expression of (LETM1)n restored growth of the yol027Δ mutant, although not as well as expression of (YOL027)n. Apparently, LetM1p is targeted to the yeast mitochondria and can functionally replace its Yol027 homolog. The yeast homolog YPR125, expressed from a multicopy plasmid, also restored growth of the yol027Δ mutant strain (Fig. 4). Mg2+ and Ca2+ Influx into Wild-type and Mutant yol027Δ Mitochondria—Partial suppression of the mrs2Δ phenotype by (YOL027)n or by (YPR125)n suggested to us that these two proteins might be involved in mitochondrial cation homeostasis. Comparing influx of Mg2+ and Ca2+ into isolated mitochondria (3Kolisek M. Zsurka G. Samaj J. Weghuber J. Schweyen R.J. Schweigel M. EMBO J. 2003; 22: 1235-1244Crossref PubMed Scopus (170) Google Scholar), we observed a considerably reduced influx of both Mg2+ and Ca2+ into mutant yol027Δ mitochondria as compared with wild-type mitochondria (Fig. 5, A and B). Although an increase in external Mg2+ or Ca2+ elicited an initial rapid response, influx quickly ceased, leading to steady-state plateau levels considerably lower in yol027Δ than in wild-type mitochondria (Fig. 5, A and B). This result indicates to us that the Mg2+ and Ca2+ transport systems are active, but saturation of influx is reached at comparatively low intramitochondrial cation concentrations. As shown by Ref. 3Kolisek M. Zsurka G. Samaj J. Weghuber J. Schweyen R.J. Schweigel M. EMBO J. 2003; 22: 1235-1244Crossref PubMed Scopus (170) Google Scholar, the driving force for Mg2+ uptake by Mrs2p is the internally negative membrane potential Δψ of about –150 mV in mitochondria. We speculated that the absence of Yol027p might result in a reduced Δψ and, hence, reduced Mg2+ and Ca2+ influx, whereas overexpression of Yol027p might have increased Δψ and thus improve Mg2+ influx in mrs2Δ (by so far unknown pathways). In fact, addition of the exogenous cation/H+ exchanger nigericin, which is known to enhance Δψ in respiring mitochondria, was found to stimulate Mg2+ influx into yol027Δ cells to a considerable extent (Fig. 5A). Effects of yol027Δ Mutation on Mitochondrial Δψ and K+ Concentrations—To determine the Δψ of mitochondria isolated from wild-type and mutant yol027Δ cells, we used JC-1, a fluorescent imidazole cyanine dye that stays monomeric at low Δψ, has a yellowish-green fluorescence, and aggregates with increasing Δψ, thereby shifting from green to orange-red fluorescence (23Reers M. Smiley S.T. Mottola-Hartshorn C. Chen A. Lin M. Chen L.B. Methods Enzymol. 1995; 260: 406-417Crossref PubMed Scopus (562) Google Scholar). As shown in Fig. 6a, mitochondrial preparations of wild-type yeast cells exhibit red fluorescence, which is consistent with a high Δψ. Yellow and green spots point to heterogeneity of the membrane potential among mitochondrial particles and even within particles, a phenomenon that has previously been described for mammalian mitochondria (23Reers M. Smiley S.T. Mottola-Hartshorn C. Chen A. Lin M. Chen L.B. Methods Enzymol. 1995; 260: 406-417Crossref PubMed Scopus (562) Google Scholar). I

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signalling. It is therefore tightly regulated. It has been recently proposed that LETM1 an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+-Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity.

LETM1 has a prominent function in mitochondrial K+ and Ca2+ homeostasis as the long-sought H+/cation exchanger.Under physiological conditions LETM1 provides mitochondria with a pathway for cation release.LETM1 is essential for the survival of all organisms tested so far, which highlights the importance of mitochondrial cation homeostasis for cell function.The recently resolved hexameric structure suggests that LETM1 could mediate cation exchange without the need for additional proteins. Mitochondrial function is essential for life. Therefore, it is unsurprising that perturbations in mitochondrial function have wide-ranging consequences in the cell. High-throughput screening has identified essential genes required for cellular survival and fitness. One such gene is LETM1. The undisputed function of LETM1 from yeast to human is to maintain the mitochondrial osmotic balance. Osmotic imbalance has been demonstrated to affect mitochondrial morphology, dynamics, and, more recently, metabolism. Whether conservation of osmotic homeostasis by LETM1 occurs by extrusion of excess mitochondrial potassium (K+), calcium (Ca2+), or both has been a matter of dispute over the past 10 years. In this Opinion, we report and discuss recent findings on LETM1 structure, essentiality, and function and its involvement in Wolf–Hirschhorn syndrome (WHS) and seizures. Mitochondrial function is essential for life. Therefore, it is unsurprising that perturbations in mitochondrial function have wide-ranging consequences in the cell. High-throughput screening has identified essential genes required for cellular survival and fitness. One such gene is LETM1. The undisputed function of LETM1 from yeast to human is to maintain the mitochondrial osmotic balance. Osmotic imbalance has been demonstrated to affect mitochondrial morphology, dynamics, and, more recently, metabolism. Whether conservation of osmotic homeostasis by LETM1 occurs by extrusion of excess mitochondrial potassium (K+), calcium (Ca2+), or both has been a matter of dispute over the past 10 years. In this Opinion, we report and discuss recent findings on LETM1 structure, essentiality, and function and its involvement in Wolf–Hirschhorn syndrome (WHS) and seizures. Mitochondrial volume homeostasis is essential for mitochondrial function and cellular viability and is regulated by ion fluxes between mitochondria and the cytosol [1.Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation.Biol. Rev. Camb. Philos. Soc. 1966; 41: 445-502Crossref PubMed Google Scholar, 2.Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. 1966.Biochim. Biophys. Acta. 2011; 1807: 1507-1538Crossref PubMed Scopus (175) Google Scholar]. The movement of ions between these compartments affects their steady-state concentrations, which leads to the alteration of several cellular processes. For instance, levels of potassium (K+), the most abundant cellular cation, are important for mitochondrial osmotic balance and levels of calcium (Ca2+) for bioenergetics, and both are critically involved in cell physiology, metabolism, and cell death regulation [3.Garlid K.D. Paucek P. Mitochondrial potassium transport: the K+ cycle.Biochim. Biophys. Acta. 2003; 1606: 23-41Crossref PubMed Scopus (306) Google Scholar, 4.Giorgi C. et al.The machineries, regulation and cellular functions of mitochondrial calcium.Nat. Rev. Mol. Cell Biol. 2018; 19: 713-730Crossref PubMed Scopus (344) Google Scholar, 5.Jarmuszkiewicz W. Szewczyk A. Energy-dissipating hub in muscle mitochondria: potassium channels and uncoupling proteins.Arch. Biochem. Biophys. 2019; 664: 102-109Crossref PubMed Scopus (8) Google Scholar, 6.Pallafacchina G. et al.Recent advances in the molecular mechanism of mitochondrial calcium uptake.F1000Res. 2018; 7: 1858Crossref Scopus (36) Google Scholar, 7.Yu S.P. Regulation and critical role of potassium homeostasis in apoptosis.Prog. Neurobiol. 2003; 70: 363-386Crossref PubMed Scopus (300) Google Scholar]. The LETM1 gene family share a central role in regulating mitochondrial cation transport and osmotic volume. LETM1 proteins are found in all eukaryotes and are highly conserved in amino acid sequence and protein architecture and can functionally complement each other as validated in some eukaryotic species [8.Nowikovsky K. et al.The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf–Hirschhorn syndrome.J. Biol. Chem. 2004; 279: 30307-30315Crossref PubMed Scopus (159) Google Scholar, 9.McQuibban A.G. et al.A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf–Hirschhorn syndrome.Hum. Mol. Genet. 2010; 19: 987-1000Crossref PubMed Scopus (67) Google Scholar] (Figure 1). The exact function of LETM1 in K+ and Ca2+ regulation and protein synthesis has been debated for a long time [8.Nowikovsky K. et al.The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf–Hirschhorn syndrome.J. Biol. Chem. 2004; 279: 30307-30315Crossref PubMed Scopus (159) Google Scholar, 10.Jiang D. et al.Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.Science. 2009; 326: 144-147Crossref PubMed Scopus (398) Google Scholar, 11.Frazier A.E. et al.Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery.J. Cell Biol. 2006; 172: 553-564Crossref PubMed Scopus (113) Google Scholar]. Since the field was last reviewed [12.Nowikovsky K. Bernardi P. LETM1 in mitochondrial cation transport.Front. Physiol. 2014; 5: 83Crossref PubMed Scopus (30) Google Scholar, 13.Nowikovsky K. et al.Perspectives on: SGP symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1.J. Gen. Physiol. 2012; 139: 445-454Crossref PubMed Scopus (54) Google Scholar], several articles have been published, providing evidence for a new transmembrane (TM) region [14.Lee S.Y. et al.Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells.J. Am. Chem. Soc. 2017; 139: 3651-3662Crossref PubMed Scopus (50) Google Scholar] and for the essentiality of the LETM1 protein [15.Blomen V.A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (503) Google Scholar, 16.Wang T. et al.Identification and characterization of essential genes in the human genome.Science. 2015; 350: 1096-1101Crossref PubMed Scopus (945) Google Scholar] as well as defining the structure of the protein oligomers [17.Shao J. et al.Leucine zipper–EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter.Sci. Rep. 2016; 634174Crossref PubMed Scopus (41) Google Scholar]. Initially, LETM1 was identified as an essential component of the mitochondrial K+/H+ exchanger (KHE) (see Glossary) [8.Nowikovsky K. et al.The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf–Hirschhorn syndrome.J. Biol. Chem. 2004; 279: 30307-30315Crossref PubMed Scopus (159) Google Scholar], a key player in mitochondrial K+ balance by releasing excess matrix K+. However, the recent proposal that LETM1 is the well-characterized but until then molecularly unknown mitochondrial Ca2+/H+ exchanger (CHX) sparked a controversy that is not yet settled [10.Jiang D. et al.Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.Science. 2009; 326: 144-147Crossref PubMed Scopus (398) Google Scholar, 17.Shao J. et al.Leucine zipper–EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter.Sci. Rep. 2016; 634174Crossref PubMed Scopus (41) Google Scholar, 18.Tsai M.F. et al.Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1.J. Gen. Physiol. 2014; 143: 67-73Crossref PubMed Scopus (99) Google Scholar]. The mitochondrial CHX is one of the mitochondrial Ca2+ release systems along with the mitochondrial Na+/Ca2+ exchanger (NCLX) [19.Boyman L. et al.NCLX: the mitochondrial sodium calcium exchanger.J. Mol. Cell. Cardiol. 2013; 59: 205-213Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar] and the mitochondrial permeability transition pore (PTP) [20.Giorgio V. et al.Dimers of mitochondrial ATP synthase form the permeability transition pore.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5887-5892Crossref PubMed Scopus (692) Google Scholar]. Regardless of the cation, LETM1’s role in maintaining mitochondrial osmotic balance is well appreciated and accepted as central to its function [9.McQuibban A.G. et al.A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf–Hirschhorn syndrome.Hum. Mol. Genet. 2010; 19: 987-1000Crossref PubMed Scopus (67) Google Scholar, 21.Dimmer K.S. et al.LETM1, deleted in Wolf–Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.Hum. Mol. Genet. 2008; 17: 201-214Crossref PubMed Scopus (150) Google Scholar, 22.Doonan P.J. et al.LETM1-dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation.FASEB J. 2014; 28: 4936-4949Crossref PubMed Scopus (81) Google Scholar, 23.Tang G. et al.The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity, production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum.Mol. Plant Pathol. 2018; 19: 1595-1611Crossref PubMed Scopus (22) Google Scholar, 24.Hasegawa A. van der Bliek A.M. Inverse correlation between expression of the Wolfs Hirschhorn candidate gene Letm1 and mitochondrial volume in C. elegans and in mammalian cells.Hum. Mol. Genet. 2007; 16: 2061-2071Crossref PubMed Scopus (53) Google Scholar, 25.Hashimi H. et al.Trypanosome Letm1 protein is essential for mitochondrial potassium homeostasis.J. Biol. Chem. 2013; 288: 26914-26925Crossref PubMed Scopus (47) Google Scholar]. In this Opinion, we first outline the essential functions of LETM1 and contrast the new protein structures with the original models. We then discuss evidence for the role of LETM1 as a KHE and a CHX, as well as address the limitations of these studies, and ultimately propose a model that explains why LETM1 has been shown to be involved in the regulation of both cations. Finally, we underline the importance of further studies to reassess these conclusions to resolve the function of LETM1 in mitochondrial biology. Essential genes are vital for biological functions that maintain cell fitness and survival and thus are indispensable for reproduction, single-cell proliferation, organismal growth, and development [26.Rancati G. et al.Emerging and evolving concepts in gene essentiality.Nat. Rev. Genet. 2018; 19: 34-49Crossref PubMed Scopus (141) Google Scholar, 27.Zhang Z. Ren Q. Why are essential genes essential? The essentiality of Saccharomyces genes.Microb. Cell. 2015; 2: 280-287Crossref PubMed Scopus (24) Google Scholar]. Two independent articles have identified LETM1 as being among a common set of approximately 2000 essential genes [15.Blomen V.A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (503) Google Scholar, 16.Wang T. et al.Identification and characterization of essential genes in the human genome.Science. 2015; 350: 1096-1101Crossref PubMed Scopus (945) Google Scholar]. Both studies used complementary high-throughput screens that relied on either gene-trap haploid cell or CRISPR technology in haploid and diploid cells. A score was then calculated based on the effect that inactivation of a specific gene had on cell survival. The complementarity of the approaches increases confidence in the importance of LETM1 and is consistent with empirical evidence described next. Deletion of the yeast LETM1 homologue, MDM38 is lethal under nonfermentable conditions [8.Nowikovsky K. et al.The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf–Hirschhorn syndrome.J. Biol. Chem. 2004; 279: 30307-30315Crossref PubMed Scopus (159) Google Scholar]. In pathogens, FgLETM1 was vital for the virulence, growth, and germination of Fusarium graminearum [23.Tang G. et al.The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity, production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum.Mol. Plant Pathol. 2018; 19: 1595-1611Crossref PubMed Scopus (22) Google Scholar], and conditional gene knockdown in Toxoplasma gondii similarly resulted in loss of virulence, growth, invasion, and replication [28.Chang L. et al.Identification and characterization of Letm1 gene in Toxoplasma gondii.Acta Biochim. Biophys. Sin. Shanghai. 2018; 51: 78-87Crossref Scopus (3) Google Scholar]. In Drosophila, organ-specific LETM1 depletion impaired tissue development and locomotor behaviour [9.McQuibban A.G. et al.A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf–Hirschhorn syndrome.Hum. Mol. Genet. 2010; 19: 987-1000Crossref PubMed Scopus (67) Google Scholar]. Consistent with haploinsufficiency of human LETM1 [29.Endele S. et al.LETM1, a novel gene encoding a putative EF-hand Ca2+-binding protein, flanks the Wolf–Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.Genomics. 1999; 60: 218-225Crossref PubMed Scopus (122) Google Scholar, 30.Hart L. et al.LETM1 haploinsufficiency causes mitochondrial defects in cells from humans with Wolf–Hirschhorn syndrome: implications for dissecting the underlying pathomechanisms in this condition.Dis. Model. Mech. 2014; 7: 535-545Crossref PubMed Scopus (24) Google Scholar, 31.Schlickum S. et al.LETM1, a gene deleted in Wolf–Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein.Genomics. 2004; 83: 254-261Crossref PubMed Scopus (79) Google Scholar, 32.Zollino M. et al.Mapping the Wolf–Hirschhorn syndrome phenotype outside the currently accepted WHS critical region and defining a new critical region, WHSCR-2.Am. J. Hum. Genet. 2003; 72: 590-597Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar], LETM1 gene deletion in animals was unachievable, as demonstrated by developmental lethality under ubiquitous downregulation of LETM1 in flies [9.McQuibban A.G. et al.A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf–Hirschhorn syndrome.Hum. Mol. Genet. 2010; 19: 987-1000Crossref PubMed Scopus (67) Google Scholar] and worms [24.Hasegawa A. van der Bliek A.M. Inverse correlation between expression of the Wolfs Hirschhorn candidate gene Letm1 and mitochondrial volume in C. elegans and in mammalian cells.Hum. Mol. Genet. 2007; 16: 2061-2071Crossref PubMed Scopus (53) Google Scholar] and embryonic lethality (E6.5) on homozygote deletion in mice [33.Jiang D. et al.Letm1, the mitochondrial Ca2+/H+ antiporter, is essential for normal glucose metabolism and alters brain function in Wolf–Hirschhorn syndrome.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2249-E2254Crossref PubMed Scopus (90) Google Scholar]. Heterozygote mice were viable but were generated in non-Mendelian ratios, highlighting the importance of LETM1 for fitness [33.Jiang D. et al.Letm1, the mitochondrial Ca2+/H+ antiporter, is essential for normal glucose metabolism and alters brain function in Wolf–Hirschhorn syndrome.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2249-E2254Crossref PubMed Scopus (90) Google Scholar]. Together with the high-throughput data and the appreciation of haploinsufficiency as a critical feature of essentiality [34.Bartha I. et al.Human gene essentiality.Nat. Rev. Genet. 2018; 19: 51-62Crossref PubMed Scopus (113) Google Scholar], these studies further demonstrate that LETM1 is indispensable in multicellular organisms and is involved in organismal development. The importance of LETM1 in mitochondrial biology is also supported by its apparent conserved protein structure. Of note, LETM1 was originally considered to contain a single TM domain [13.Nowikovsky K. et al.Perspectives on: SGP symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1.J. Gen. Physiol. 2012; 139: 445-454Crossref PubMed Scopus (54) Google Scholar, 21.Dimmer K.S. et al.LETM1, deleted in Wolf–Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.Hum. Mol. Genet. 2008; 17: 201-214Crossref PubMed Scopus (150) Google Scholar], although recent data suggest the existence of a second TM region; this may have important implications in LETM1’s protein topology and interaction with other proteins [14.Lee S.Y. et al.Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells.J. Am. Chem. Soc. 2017; 139: 3651-3662Crossref PubMed Scopus (50) Google Scholar] (Box 1). Furthermore, novel 3D data proposed a model of a hexameric structure for recombinant LETM1 with a central pore that can be regulated by pH [17.Shao J. et al.Leucine zipper–EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter.Sci. Rep. 2016; 634174Crossref PubMed Scopus (41) Google Scholar]. However, these data are still in need of further validation, as explained in this Opinion.Box 1LETM1 Protein Architecture: Evidence of a New TopologyThe LETM1 family of proteins have a predominantly conserved structure [12.Nowikovsky K. Bernardi P. LETM1 in mitochondrial cation transport.Front. Physiol. 2014; 5: 83Crossref PubMed Scopus (30) Google Scholar]. The most notable feature is the LETM1-like region (PFAM: PF07766) [66.Marchler-Bauer A. et al.CDD: NCBI’s Conserved Domain Database.Nucleic Acids Res. 2015; 43: D222-D226Crossref PubMed Scopus (2257) Google Scholar]. The original consensus was that LETM1 proteins have a single TM helix, one 14-3-3-like ribosome-binding domain (RBD) [58.Gunter T.E. Pfeiffer D.R. Mechanisms by which mitochondria transport calcium.Am. J. Phys. 1990; 258: C755-C786Crossref PubMed Google Scholar], several coiled-coil domains, and one or two EF-hand motifs [13.Nowikovsky K. et al.Perspectives on: SGP symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1.J. Gen. Physiol. 2012; 139: 445-454Crossref PubMed Scopus (54) Google Scholar], except fungi, which lack the EF-hands (Figure I). Unlike the EF-hands of mitochondrial carriers or MCU regulator proteins, which face the IMS, the presence of an EF-hand on the matrix side of LETM1 [35.Hajnoczky G. et al.Reliance of ER–mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins: Miros, MICUs, LETM1 and solute carriers.Curr. Opin. Cell Biol. 2014; 29: 133-141Crossref PubMed Scopus (34) Google Scholar] suggested a matrix-Ca2+-sensing role. Given the predicted single-TM nature of the protein, early studies determined the topology of the protein to be Cin, Nout [21.Dimmer K.S. et al.LETM1, deleted in Wolf–Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.Hum. Mol. Genet. 2008; 17: 201-214Crossref PubMed Scopus (150) Google Scholar]. However, Lee et al. in their recent topology-mapping analyses have proposed a new model that may change the perspective of LETM1 being a single-TM protein [13.Nowikovsky K. et al.Perspectives on: SGP symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1.J. Gen. Physiol. 2012; 139: 445-454Crossref PubMed Scopus (54) Google Scholar, 21.Dimmer K.S. et al.LETM1, deleted in Wolf–Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.Hum. Mol. Genet. 2008; 17: 201-214Crossref PubMed Scopus (150) Google Scholar]. Combining a peroxidase-sensitive desthiobiotin-phenol probe, which labels tyrosine residues using phenoxyl radicals, with mass spectrometric analysis of modified peptides, Lee et al. have uncovered a second possible TM that none of the prediction algorithms (TMHMM and TMpred) has to date suggested [14.Lee S.Y. et al.Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells.J. Am. Chem. Soc. 2017; 139: 3651-3662Crossref PubMed Scopus (50) Google Scholar]. The newly defined topology of LETM1 determines two TM regions that place both protein terminals in the matrix [14.Lee S.Y. et al.Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells.J. Am. Chem. Soc. 2017; 139: 3651-3662Crossref PubMed Scopus (50) Google Scholar]. There is a possibility of the N-terminal residues containing the mitochondrial translocating sequence (MTS) being inadvertently tagged during membrane insertion. The MTS must be cleaved in the matrix, therefore providing an opportunity for tagging of these residues before this N-terminal portion is flipped into the IMS. The use of a bulky tag for such an intricate analysis is also questionable as it belies the physiological relevance of the mutational analysis. At closer view, the data on protease protection of isolated mitochondria of Shao et al. do not appear in conflict with the new topology [17.Shao J. et al.Leucine zipper–EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter.Sci. Rep. 2016; 634174Crossref PubMed Scopus (41) Google Scholar]. They show that the full-length LETM1 was protected by the inner membrane from cleavage like matrix PRX3 and was more resistant to cleavage than the inner-membrane TIM23. However, further detailed mapping studies are still needed. Once reliably clarified, these new data are of great relevance as they change the topology of several domains and modified amino acids and will then have to be considered in future functional and structural studies of LETM1. The LETM1 family of proteins have a predominantly conserved structure [12.Nowikovsky K. Bernardi P. LETM1 in mitochondrial cation transport.Front. Physiol. 2014; 5: 83Crossref PubMed Scopus (30) Google Scholar]. The most notable feature is the LETM1-like region (PFAM: PF07766) [66.Marchler-Bauer A. et al.CDD: NCBI’s Conserved Domain Database.Nucleic Acids Res. 2015; 43: D222-D226Crossref PubMed Scopus (2257) Google Scholar]. The original consensus was that LETM1 proteins have a single TM helix, one 14-3-3-like ribosome-binding domain (RBD) [58.Gunter T.E. Pfeiffer D.R. Mechanisms by which mitochondria transport calcium.Am. J. Phys. 1990; 258: C755-C786Crossref PubMed Google Scholar], several coiled-coil domains, and one or two EF-hand motifs [13.Nowikovsky K. et al.Perspectives on: SGP symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1.J. Gen. Physiol. 2012; 139: 445-454Crossref PubMed Scopus (54) Google Scholar], except fungi, which lack the EF-hands (Figure I). Unlike the EF-hands of mitochondrial carriers or MCU regulator proteins, which face the IMS, the presence of an EF-hand on the matrix side of LETM1 [35.Hajnoczky G. et al.Reliance of ER–mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins: Miros, MICUs, LETM1 and solute carriers.Curr. Opin. Cell Biol. 2014; 29: 133-141Crossref PubMed Scopus (34) Google Scholar] suggested a matrix-Ca2+-sensing role. Given the predicted single-TM nature of the protein, early studies determined the topology of the protein to be Cin, Nout [21.Dimmer K.S. et al.LETM1, deleted in Wolf–Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.Hum. Mol. Genet. 2008; 17: 201-214Crossref PubMed Scopus (150) Google Scholar]. However, Lee et al. in their recent topology-mapping analyses have proposed a new model that may change the perspective of LETM1 being a single-TM protein [13.Nowikovsky K. et al.Perspectives on: SGP symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1.J. Gen. Physiol. 2012; 139: 445-454Crossref PubMed Scopus (54) Google Scholar, 21.Dimmer K.S. et al.LETM1, deleted in Wolf–Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.Hum. Mol. Genet. 2008; 17: 201-214Crossref PubMed Scopus (150) Google Scholar]. Combining a peroxidase-sensitive desthiobiotin-phenol probe, which labels tyrosine residues using phenoxyl radicals, with mass spectrometric analysis of modified peptides, Lee et al. have uncovered a second possible TM that none of the prediction algorithms (TMHMM and TMpred) has to date suggested [14.Lee S.Y. et al.Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells.J. Am. Chem. Soc. 2017; 139: 3651-3662Crossref PubMed Scopus (50) Google Scholar]. The newly defined topology of LETM1 determines two TM regions that place both protein terminals in the matrix [14.Lee S.Y. et al.Architecture mapping of the inner mitochondrial membrane proteome by chemical tools in live cells.J. Am. Chem. Soc. 2017; 139: 3651-3662Crossref PubMed Scopus (50) Google Scholar]. There is a possibility of the N-terminal residues containing the mitochondrial translocating sequence (MTS) being inadvertently tagged during membrane insertion. The MTS must be cleaved in the matrix, therefore providing an opportunity for tagging of these residues before this N-terminal portion is flipped into the IMS. The use of a bulky tag for such an intricate analysis is also questionable as it belies the physiological relevance of the mutational analysis. At closer view, the data on protease protection of isolated mitochondria of Shao et al. do not appear in conflict with the new topology [17.Shao J. et al.Leucine zipper–EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter.Sci. Rep. 2016; 634174Crossref PubMed Scopus (41) Google Scholar]. They show that the full-length LETM1 was protected by the inner membrane from cleavage like matrix PRX3 and was more resistant to cleavage than the inner-membrane TIM23. However, further detailed mapping studies are still needed. Once reliably clarified, these new data are of great relevance as they change the topology of several domains and modified amino acids and will then have to be considered in future functional and structural studies of LETM1. Although there is evolutionary conservation of LETM1 proteins, the presence and number of EF-hand motifs varies across organisms. EF-hands mediate Ca2+ sensing, and mitochondrial EF-hand-containing proteins play a pivotal role in sensing Ca2+ to facilitate mitochondrial Ca2+ buffering [35.Hajnoczky G. et al.Reliance of ER–mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins: Miros, MICUs, LETM1 and solute carriers.Curr. Opin. Cell Biol. 2014; 29: 133-141Crossref PubMed Scopus (34) Google Scholar]. Therefore, among mitochondrial EF-hand-containing proteins [35.Hajnoczky G. et al.Reliance of ER–mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins: Miros, MICUs, LETM1 and solute carriers.Curr. Opin. Cell Biol. 2014; 29: 133-141Crossref PubMed Scopus (34) Google Scholar], it is surprising that only LETM1, mitochondrial Rho GTPase (MIRO), and mitochondrial calcium uptake 1 (MICU1) were identified as essential across several cell lines in genome-wide essentiality screens [15.Blomen V.A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (503) Google Scholar, 16.Wang T. et al.Identification and characterization of essential genes in the human genome.Science. 2015; 350: 1096-1101Crossref PubMed Scopus (945) Google Scholar]. None of the major regulators of mitochondrial Ca2+ without EF-hands, such as the NCLX, mitochondrial calcium uptake (MCU), or the components of the core MCU complex, MCUb and essential MCU regulator (EMRE) [36.Sancak Y. et al.EMRE is an essential component of the mitochondrial calcium uniporter complex.Science. 2013; 342: 1379-1382Crossref PubMed Scopus (444) Google Scholar], were identified as essential in either screen. As these proteins are critical for mitochondrial Ca2+ homeostasis [4.Giorgi C. et al.The machineries, regulation and cellular functions of mitochondrial calcium.Nat. Rev. Mol. Cell Biol. 2018; 19: 713-730Crossref PubMed Scopus (344) Google Scholar], which is crucial for mitochondrial function, they would be expected to be vital. However, their function is likely to be regulated in complex ways or backed-up by redundant proteins. Furthermore, they may be synthetic lethal with other mitochondrial transporters or regulators [15.Blomen V.A. et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (503) Google Scholar] or, like NCLX, are conditional lethal [37.Luongo T.S. et al.The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.Nature. 2017; 545: 93-97Crossref PubMed Scopus (226) Google Scholar]. LETM1 was originally identified as a gene candidate for seizures in the genetic disorder Wolf Hirschhorn syndrome (WHS) (Box 2), most likely perturbing mitochondrial function [29.Endele S. et al.LETM1, a novel gene encoding a putative EF-hand Ca2+-binding protein, flanks the Wolf–Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.Genomics. 1999; 60: 218-225Crossref PubMed Scopus (122) Google Scholar, 31.Schlickum S. et al.LETM1, a gene deleted in Wolf–Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein.Genomics. 2004; 83: 254-261Crossref PubMed Scopus (79) Google Scholar, 32.Zollino M. et al.Mapping the Wolf–Hirschhorn syndrome phenotype outside the currently accepted WHS critical region and defining a new critical region, WHSCR-2.Am. J. Hum. Genet. 2003; 72: 590-597Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar]. Concurrently, MDM38 appeared in a genome-wide screen for genes involved in mitochondrial distribution and morphology [38.Dimmer K.S. et al.Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae.Mol. Biol. Cell. 2002; 13: 847-853Crossref PubMed Scopus (348) Google Scholar]. Mitochondrial fragmentation, matrix swelling, and

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.

Regulation of mitochondrial volume is a key issue in cellular pathophysiology. Mitochondrial volume and shape changes can occur following regulated fission–fusion events, which are modulated by a complex network of cytosolic and mitochondrial proteins; and through regulation of ion transport across the inner membrane. In this review we will cover mitochondrial volume homeostasis that depends on (i) monovalent cation transport across the inner membrane, a regulated process that couples electrophoretic K+ influx on K+ channels to K+ extrusion through the K+–H+ exchanger; (ii) the permeability transition, a loss of inner membrane permeability that may be instrumental in triggering cell death. Specific emphasis will be placed on molecular advances on the nature of the transport protein(s) involved, and/or on diseases that depend on mitochondrial volume dysregulation.

Originally identified as a key element of mitochondrial volume homeostasis through regulation of K+–H+ exchange (KHE), the LETM1 protein family is also involved in respiratory chain biogenesis and in the pathogenesis of seizures in the Wolf–Hirschhorn syndrome (WHS). To add further complexity,

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me2NNMe2 differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me2NNMe2- (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me2NNMe2 (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca2+ and ER thiol redox homeostasis. Our findings indicate that compounds like Me2NNMe2 are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.

YOL027c in yeast and LETM1 in humans encode integral proteins of the inner mitochondrial membrane. They have been implicated in mitochondrial K+ homeostasis and volume control. To further characterize their role, we made use of submitochondrial particles (SMPs) with entrapped K+- and H+-sensitive fluorescent dyes PBFI and BCECF, respectively, to study the kinetics of K+ and H+ transport across the yeast inner mitochondrial membrane. Wild-type SMPs exhibited rapid, reciprocal translocations of K+ and H+ driven by concentration gradients of either of them. K+ and H+ translocations have stoichiometries similar to those mediated by the exogenous K+/H+ exchanger nigericin, and they are shown to be essentially electroneutral and obligatorily coupled. Moreover, [K+] gradients move H+ against its concentration gradient, and vice-versa. These features, as well as the sensitivity of K+ and H+ fluxes to quinine and Mg2+, qualify these activities as K+/H+ exchange reactions. Both activities are abolished when the yeast Yol027p protein is absent (yol027Delta mutant SMPs), indicating that it has an essential role in this reaction. The replacement of the yeast Yol027p by the human Letm1 protein restores K+/H+ exchange activity confirming functional homology of the yeast and human proteins. Considering their newly identified function, we propose to refer to the yeast YOL027c gene and the human LETM1 gene as yMKH1 and hMKH1, respectively.

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling, and autophagic decay of the organelle. We have previously identified the yeast Mdm38 and its human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn syndrome, as essential components of the KHE. In a genome-wide screen for multicopy suppressors of the pet(-) (reduced growth on nonfermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate suppressors and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner membrane proteins and constituents of approximately 500-kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential, and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp, or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Based on these results, we propose that Ydl183p and the Mdm38p homologues Mrs7p, LETM1, and HCCR-1 are involved in the formation of an active KHE system.

The leading first-in-class ruthenium-complex BOLD-100 currently undergoes clinical phase-II anticancer evaluation. Recently, BOLD-100 is identified as anti-Warburg compound. The present study shows that also deregulated lipid metabolism parameters characterize acquired BOLD-100-resistant colon and pancreatic carcinoma cells. Acute BOLD-100 treatment reduces lipid droplet contents of BOLD-100-sensitive but not -resistant cells. Despite enhanced glycolysis fueling lipid accumulation, BOLD-100-resistant cells reveal diminished lactate secretion based on monocarboxylate transporter 1 (MCT1) loss mediated by a frame-shift mutation in the MCT1 chaperone basigin. Glycolysis and lipid catabolism converge in the production of protein/histone acetylation substrate acetyl-coenzymeA (CoA). Mass spectrometric and nuclear magnetic resonance analyses uncover spontaneous cell-free BOLD-100-CoA adduct formation suggesting acetyl-CoA depletion as mechanism bridging BOLD-100-induced lipid metabolism alterations and histone acetylation-mediated gene expression deregulation. Indeed, BOLD-100 treatment decreases histone acetylation selectively in sensitive cells. Pharmacological targeting confirms histone de-acetylation as central mode-of-action of BOLD-100 and metabolic programs stabilizing histone acetylation as relevant Achilles' heel of acquired BOLD-100-resistant cell and xenograft models. Accordingly, histone gene expression changes also predict intrinsic BOLD-100 responsiveness. Summarizing, BOLD-100 is identified as epigenetically active substance acting via targeting several onco-metabolic pathways. Identification of the lipid metabolism as driver of acquired BOLD-100 resistance opens novel strategies to tackle therapy failure.

GENERAL COMMENTARY article Front. Physiol., 26 February 2014Sec. Mitochondrial Research https://doi.org/10.3389/fphys.2014.00083

This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46.KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics.Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme.Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy.Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization.Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L.Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway.

On the basis of enhanced tumor accumulation and bone affinity, gallium compounds are under development as anticancer and antimetastatic agents. In this study, we analyzed molecular targets of one of the lead anticancer gallium complexes [KP46, Tris(8-quinolinolato)gallium(III)] focusing on colon and lung cancer. Within a few hours, KP46 treatment at low micromolar concentrations induced cell body contraction and loss of adhesion followed by prompt cell decomposition. This rapid KP46-induced cell death lacked classic apoptotic features and was insensitive toward a pan-caspase inhibitor. Surprisingly, however, it was accompanied by upregulation of proapoptotic Bcl-2 family members. Furthermore, a Bax- but not a p53-knockout HCT-116 subline exhibited significant KP46 resistance. Rapid KP46-induced detachment was accompanied by downregulation of focal adhesion proteins, including several integrin subunits. Loss of integrin-β1 and talin plasma membrane localization corresponded to reduced binding of RGD (Arg-Gly-Asp) peptides to KP46-treated cells. Accordingly, KP46-induced cell death and destabilization of integrins were enhanced by culture on collagen type I, a major integrin ligand. In contrast, KP46-mediated adhesion defects were partially rescued by Mg(2+) ions, promoting integrin-mediated cell adhesion. Focal adhesion dynamics are regulated by calpains via cleavage of multiple cell adhesion molecules. Cotreatment with the cell-permeable calpain inhibitor PD150606 diminished KP46-mediated integrin destabilization and rapid cell death induction. KP46 treatment distinctly inhibited HCT-116 colon cancer xenograft in vivo by causing reduced integrin plasma membrane localization, tissue disintegration, and intense tumor necrosis. This study identifies integrin deregulation via a calpain-mediated mechanism as a novel mode of action for the anticancer gallium compound KP46.

There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction.

This review provides a retrospective on the role of osmotic regulation in the process of eukaryogenesis. Specifically, it focuses on the adjustments which must have been made by the original colonizing α-proteobacteria that led to the evolution of modern mitochondria. We focus on the cations that are fundamentally involved in volume determination and cellular metabolism and define the transporter landscape in relation to these ions in mitochondria as we know today. We provide analysis on how the cations interplay and together maintain osmotic balance that allows for effective ATP synthesis in the organelle.

Cadmium (Cd) accumulates with aging and is elevated in long-lived species. Metallothioneins (MTs), small cysteine-rich proteins involved in metal homeostasis and Cd detoxification, are known to be related to longevity. However, the relationship between Cd accumulation, the role of MTs, and aging is currently unclear. Specifically, we do not know if long-lived species evolved an efficient metal stress response by upregulating their MT levels to reduce the toxic effects of environmental pollutants, such as Cd, that accumulate over their longer life span. It is also unknown if the number of MT genes, their expression, or both protect the organisms from potentially damaging effects during aging. To address these questions, we reanalyzed several cross-species studies and obtained data on MT expression and Cd accumulation in long-lived mouse models. We confirmed a relationship between species maximum life span in captive mammals and their Cd content in liver and kidney. We found that although the number of MT genes does not affect longevity, gene expression and protein amount of specific MT paralogs are strongly related to life span in mammals. MT expression rather than gene number may influence the high Cd levels and longevity of some species. In support of this, we found that overexpression of MT-1 accelerated Cd accumulation in mice and that tissue Cd was higher in long-lived mouse strains with high MT expression. We conclude that long-lived species have evolved a more efficient stress response by upregulating the expression of MT genes in presence of Cd, which contributes to elevated tissue Cd levels.

Mass spectrometric-based proteomics is a powerful tool to analyze post-translationally modified proteins. Carbonylation modifications that result from oxidative lipid breakdown are a class of post-translational modifications that are poorly characterized with respect to protein targets and function. This is partly due to the lack of dedicated mass spectrometry-based technologies to facilitate the analysis of these modifications. Here, we present a comprehensive approach to identify malondialdehyde-modified proteins and peptides. Malondialdehyde is among the most abundant of the lipid peroxidation products; and malondialdehyde-derived adducts on proteins have been implicated in cardiovascular diseases, neurodegenerative disorders, and other clinical conditions. Our integrated approach targets three levels of the overall proteomic workflow: (i) sample preparation, by employing a targeted enrichment strategy; (ii) high-performance liquid chromatography, by using a gradient optimized for the separation of the modified peptides; and (iii) tandem mass spectrometry, by improving the spectral quality of very low-abundance peptides. By applying the optimized procedure to a whole cell lysate spiked with a low amount of malondialdehyde-modified proteins, we were able to identify up to 350 different modified peptides and localize the modification to a specific lysine residue. This methodology allows the comprehensive analysis of malondialdehyde-modified proteins.

The interactions between apoptotic and autophagic proteins via the proteolytic systems are known mechanisms through which autophagy and apoptosis regulate each other. In this issue of The FEBS Journal, Gentle and colleagues propose a mechanism through which autophagy regulates the induction of apoptosis at the level of the TIR-domain-containing adaptor-inducing interferon-β (TRIF) in TLR signaling.

Abstract We have monitored the effects of KLKL 5 KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide‐treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide‐treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid‐induced random coil to β‐sheet and β‐sheet to α‐helix conformational transitions of KLK. Together with the oligonucleotide oligo‐d(IC) 13 [ODN1a], KLK functions as a novel adjuvant, termed IC31™. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLK's interaction with the cell surface and other membrane‐bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31™.

Mitochondria are fundamental for life and require balanced ion exchange to maintain proper functioning. The mitochondrial cation exchanger LETM1 sparks interest because of its pathophysiological role in seizures in the Wolf Hirschhorn Syndrome (WHS). Despite observation of sleep disorganization in epileptic WHS patients, and growing studies linking mitochondria and epilepsy to circadian rhythms, LETM1 has not been studied from the chronobiological perspective. Here we established a viable letm1 knock-out, using the diurnal vertebrate Danio rerio to study the metabolic and chronobiological consequences of letm1 deficiency. We report diurnal rhythms of Letm1 protein levels in wild-type fish. We show that mitochondrial nucleotide metabolism is deregulated in letm1-/- mutant fish, the rate-limiting enzyme of NAD+ production is up-regulated, while NAD+ and NADH pools are reduced. These changes were associated with increased expression amplitude of circadian core clock genes in letm1-/- compared with wild-type under light/dark conditions, suggesting decreased NAD(H) levels as a possible mechanism for circadian system perturbation in Letm1 deficiency. Replenishing NAD pool may ameliorate WHS-associated sleep and neurological disorders.

The mitochondrial K(+)/H(+) exchanger (KHE) is a key regulator of mitochondrial K(+), the most abundant cellular cation, and thus for volume control of the organelle. Downregulation of the mitochondrial KHE results in osmotic swelling and autophagic degradation of the organelle. This chapter describes methods to shut-off expression of Mdm38p, an essential factor of the mitochondrial KHE, and to observe the cellular consequences thereof, in particular changes in KHE activity and morphogenetic changes of mitochondria by applying new techniques developed in our laboratories.

Abstract Mitochondrial Ca 2+ ions are crucial regulators of bioenergetics, cell death pathways and cytosolic Ca 2+ homeostasis. Mitochondrial Ca 2+ content strictly depends on Ca 2+ transporters. In recent decades, the major players responsible for mitochondrial Ca 2+ uptake and release have been identified, except the mitochondrial Ca 2+ /H + exchanger (CHE). Originally identified as the mitochondrial K + /H + exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identified MICS1, the only mitochondrial member of the TMBIM family. Applying cell-based and cell-free biochemical assays, here we demonstrate that MICS1 is responsible for the Na + - and permeability transition pore-independent mitochondrial Ca 2+ release and identify MICS1 as the long-sought mitochondrial CHE. This finding provides the final piece of the puzzle of mitochondrial Ca 2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca 2+ exchange.

<div>Abstract<p>On the basis of enhanced tumor accumulation and bone affinity, gallium compounds are under development as anticancer and antimetastatic agents. In this study, we analyzed molecular targets of one of the lead anticancer gallium complexes [KP46, Tris(8-quinolinolato)gallium(III)] focusing on colon and lung cancer. Within a few hours, KP46 treatment at low micromolar concentrations induced cell body contraction and loss of adhesion followed by prompt cell decomposition. This rapid KP46-induced cell death lacked classic apoptotic features and was insensitive toward a pan–caspase inhibitor. Surprisingly, however, it was accompanied by upregulation of proapoptotic Bcl-2 family members. Furthermore, a Bax- but not a p53-knockout HCT-116 subline exhibited significant KP46 resistance. Rapid KP46-induced detachment was accompanied by downregulation of focal adhesion proteins, including several integrin subunits. Loss of integrin-β1 and talin plasma membrane localization corresponded to reduced binding of RGD (Arg–Gly–Asp) peptides to KP46-treated cells. Accordingly, KP46-induced cell death and destabilization of integrins were enhanced by culture on collagen type I, a major integrin ligand. In contrast, KP46-mediated adhesion defects were partially rescued by Mg<sup>2+</sup> ions, promoting integrin-mediated cell adhesion. Focal adhesion dynamics are regulated by calpains via cleavage of multiple cell adhesion molecules. Cotreatment with the cell-permeable calpain inhibitor PD150606 diminished KP46-mediated integrin destabilization and rapid cell death induction. KP46 treatment distinctly inhibited HCT-116 colon cancer xenograft <i>in vivo</i> by causing reduced integrin plasma membrane localization, tissue disintegration, and intense tumor necrosis. This study identifies integrin deregulation via a calpain-mediated mechanism as a novel mode of action for the anticancer gallium compound KP46. <i>Mol Cancer Ther; 13(10); 2436–49. ©2014 AACR</i>.</p></div>

<p>Supplementary Figure 1. Role of p53 status and mitochondrial membrane depolarization in KP46-induced cell death Supplementary Figure 2. Expression and localization changes of pro- and anti-apoptotic Bcl-2 family members in response to KP46 treatment Supplementary Figure 3. Impact or KP46 on integrin β1 expression and subcellular localization in cancer cells Supplementary Figure 4. Alteration in intracellular Ca2+ levels in HCT-116 cells upon KP46 treatment Supplementary Figure 5 Effect of extracellular calpain inhibition on KP46 sensitivity in A427 and HCT-116 cells</p>

<p>Supplementary Video 1. Impact of calpain inhibition on the dynamics of KP64-induced rapid cell death of A427 cells</p>

<p>Legends to all supplementary figures and the video</p>

<p>Supplementary Video 1. Impact of calpain inhibition on the dynamics of KP64-induced rapid cell death of A427 cells</p>

<p>Legends to all supplementary figures and the video</p>

<p>Supplementary Figure 1. Role of p53 status and mitochondrial membrane depolarization in KP46-induced cell death Supplementary Figure 2. Expression and localization changes of pro- and anti-apoptotic Bcl-2 family members in response to KP46 treatment Supplementary Figure 3. Impact or KP46 on integrin β1 expression and subcellular localization in cancer cells Supplementary Figure 4. Alteration in intracellular Ca2+ levels in HCT-116 cells upon KP46 treatment Supplementary Figure 5 Effect of extracellular calpain inhibition on KP46 sensitivity in A427 and HCT-116 cells</p>

<div>Abstract<p>On the basis of enhanced tumor accumulation and bone affinity, gallium compounds are under development as anticancer and antimetastatic agents. In this study, we analyzed molecular targets of one of the lead anticancer gallium complexes [KP46, Tris(8-quinolinolato)gallium(III)] focusing on colon and lung cancer. Within a few hours, KP46 treatment at low micromolar concentrations induced cell body contraction and loss of adhesion followed by prompt cell decomposition. This rapid KP46-induced cell death lacked classic apoptotic features and was insensitive toward a pan–caspase inhibitor. Surprisingly, however, it was accompanied by upregulation of proapoptotic Bcl-2 family members. Furthermore, a Bax- but not a p53-knockout HCT-116 subline exhibited significant KP46 resistance. Rapid KP46-induced detachment was accompanied by downregulation of focal adhesion proteins, including several integrin subunits. Loss of integrin-β1 and talin plasma membrane localization corresponded to reduced binding of RGD (Arg–Gly–Asp) peptides to KP46-treated cells. Accordingly, KP46-induced cell death and destabilization of integrins were enhanced by culture on collagen type I, a major integrin ligand. In contrast, KP46-mediated adhesion defects were partially rescued by Mg<sup>2+</sup> ions, promoting integrin-mediated cell adhesion. Focal adhesion dynamics are regulated by calpains via cleavage of multiple cell adhesion molecules. Cotreatment with the cell-permeable calpain inhibitor PD150606 diminished KP46-mediated integrin destabilization and rapid cell death induction. KP46 treatment distinctly inhibited HCT-116 colon cancer xenograft <i>in vivo</i> by causing reduced integrin plasma membrane localization, tissue disintegration, and intense tumor necrosis. This study identifies integrin deregulation via a calpain-mediated mechanism as a novel mode of action for the anticancer gallium compound KP46. <i>Mol Cancer Ther; 13(10); 2436–49. ©2014 AACR</i>.</p></div>

For patients with advanced stage epithelial ovarian cancer (EOC), substantial emphasis has been placed on diagnostic tests that can discern which of two treatment options – primary cytoreductive surgery (PCS) or neoadjuvant chemotherapy followed by interval cytoreductive surgery (NACT + ICS) – optimizes patient-level outcomes. Our goal was to project potential life expectancy (LE) gains that could be achieved by use of such a test.We developed a microsimulation model to project LE for patients with stage IIIC EOC. We compared: a “standard-of-care” strategy, in which patients were triaged to PCS vs. NACT + ICS based on current clinical practice; and a “test” strategy, in which patients were triaged based on results of a hypothetical test. We identified those test performance characteristics for which the test strategy outperformed the standard-of-care strategy, from a LE standpoint. Effects of parameter uncertainty were evaluated in sensitivity analysis.Even with a perfect test, the LE gain was modest (LE with test vs. standard-of-care strategy = 67.6 vs. 66.4 months; LE gain = 1.2 months). In order to outperform the standard-of-care, the test had to have a high probability of correctly identifying “resectable” patients at PCS (i.e. those for whom complete or optimal cytoreduction would be possible); this test property was more important than correct triage of unresectable patients to NACT + ICS. Results were sensitive to the proportion of patients whose underlying disease was resectable at PCS.Diagnostic tests that are designed to triage patients with advanced stage EOC will likely have only a modest effect on LE.

Mitochondrial Ca2+ elevations enhance ATP production, but uptake must be balanced by efflux to avoid overload. Uptake is mediated by the mitochondrial Ca2+ uniporter channel complex (MCUC), and extrusion is controlled largely by the Na+/Ca2+ exchanger (NCLX), both driven electrogenically by the inner membrane potential (ΔΨm). MCUC forms hotspots at the cardiac mitochondria-junctional SR (jSR) association to locally receive Ca2+ signals; however, the distribution of NCLX is unknown. Our fractionation-based assays reveal that extensively jSR-associated mitochondrial segments contain a minor portion of NCLX and lack Na+-dependent Ca2+ extrusion. This pattern is retained upon in vivo NCLX overexpression, suggesting extensive targeting to non-jSR-associated submitochondrial domains and functional relevance. In cells with non-polarized MCUC distribution, upon NCLX overexpression the same given increase in matrix Ca2+ expends more ΔΨm. Thus, cardiac mitochondrial Ca2+ uptake and extrusion are reciprocally polarized, likely to optimize the energy efficiency of local calcium signaling in the beating heart.

This paper reports quinoline-based BODIPYs as potential EGFR/VEGFR-2 inhibitors and their anticancer activities against Hela cells. Lipinski's drug likeness of compounds 1-3 was predicted revealing that they might exhibit promising physicochemical properties for oral bioavailability. The HOMO and LUMO energies were also calculated using DFT/RCAM-B3LYP method at CC-pVTZ. The EGFR/VEGFR-2 interaction was examined by molecular docking, suggesting that all compounds fitted into the pocket of VEGFR-2 within the key residues- Glu885, Cys919 and Asp1046. The binding energies calculated were in the order 3 ˃ 2 ˃ 1. The results suggested a greater binding affinity of VEGFR-2 in comparison to EGFR. The in vitro anticancer activity of the compounds 1 and 3 on the HeLa cells was evaluated, revealing significant reduction in cell viability as compared to control.

Membrane fusion is the first essential step in HIV-1 replication. The gp41 subunit of HIV-1 envelope protein (Env), a class I fusion protein, achieves membrane fusion by forming a structure called a six-helix bundle composed of N- and C-terminal heptad repeats. We have recently shown that the distal portion of the α9 helix in the C-terminal heptad repeat of X4-tropic HXB2 Env plays a critical role in the late-stage membrane fusion and viral infection. Here, we used R5-tropic JRFL Env and constructed six alanine insertion mutants, 641+A to 646+A, in the further distal portion of α9 where several glutamine residues are conserved (the number corresponds to the position of the inserted alanine in JRFL Env). 644+A showed the most severe defect in syncytia formation. Decreased fusion pore formation activity, revealed by a dual split protein assay, was observed in all mutants except 641+A. Sequence analysis and substitution of inserted 644A with Gln revealed that the glutamine residue at position 644 that forms complex hydrogen-bond networks with other polar residues on the surface of the six-helix bundle is critical for cell–cell fusion. We also developed a split NanoLuc® (Nluc) reporter-based assay specific to the virus–cell membrane fusion step to analyze several of the mutants. Interestingly syncytia-competent mutants failed to display Nluc activities. In addition to defective fusion activity, a reduction of Env incorporation into virions may further contribute to differences in cell–cell and virus–cell fusions.

Kaiyrzhanov et al. describe 18affected individuals with bi-allelic variants in the leucine zipper-EFhand containing transmembrane protein 1 gene presenting with clinical features suggestive of a mitochondrial disease.Functional studies showed defective mitochondrial K þ efflux, swollen mitochondrial matrix structures, and loss of mitochondrial oxidative phosphorylation protein components.

Abstract Acquired therapy resistance of diverse cancer types frequently involves changed metabolic processes linked to altered cellular lipid uptake or de novo synthesis. In this study, colon and pancreatic cancer cells were selected for resistance to the lead ruthenium-anticancer complex sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339), currently in clinical development. KP1339 binds serum proteins and selectively accumulates in malignant tissue. This is reflected by mild adverse effects and a promising therapeutic window. However, potential resistance mechanisms against KP1339 are enigmatic so far. HCT116 and Capan-1 cell models with acquired KP1339 resistance were established and investigated for altered gene expression profiles. mRNA data were confirmed on the protein level using immunoblotting. Lipid storage compartments (lipid droplets) were visualized fluorescently with Bodipy 493/503. The role of altered lipid metabolism components in KP1339 resistance was dissected utilizing specific pharmacological inhibitors. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed by Seahorse XF analyses. Lipidomics and consecutive proteomics analysis were used to further characterize the resistance phenotype. Resistance in both cell models was not based on a drug accumulation defect. In-depth bioinformatic analyses revealed major changes in the lipid metabolism associated with KP1339 resistance. Consistently, KP1339-resistant cells contained elevated lipid droplet levels. Hence, resistant cell models were hypersensitive towards the lipid-modulating agents triacsin C, statins, and orlistat. Unexpectedly, most of these inhibitors exerted even antagonistic activity when combined with KP1339 in parental and resistant cell models. In contrast, the ß-oxidation inhibitor etomoxir massively synergized with KP1339 and completely reverted acquired resistance. In the Seahorse Mito Stress Test, KP1339 severely reduced ECAR in sensitive HCT116 cells suggesting interference with glycolysis. Accordingly, KP1339-resistant cells exhibited clearly reduced spontaneous ECAR levels which, in contrast to the parental line, did not increase upon respiration inhibition by oligomycin. Upon KP1339 treatment, OCR was reduced in parental but stayed unchanged in resistant cells. Lipidomics confirmed distinctly enhanced lipid droplet component levels (e.g. triglycerides) associated with KP1339 resistance while proteomics indicated degradation of monocarboxylate transporters MCT-1/MCT-4. In this study, we uncover an impact on lipid metabolism as vital player in response to and acquired resistance against the anticancer ruthenium compound KP1339. We suggest the development of rational combination schemes between lipid metabolism modulators, like etomoxir, and KP1339 for enhanced activity and resistance prevention. The respective preclinical in vivo experiments are currently initiated. Citation Format: Dina Salome Baier, Beatrix Schoenhacker-Alte, Christine Pirker, Bernhard Englinger, Thomas Mohr, Samuel Meier-Menches, Clemens Roehrl, Patrick Moser, Anna Laemmerer, Benjamin Neuditschko, Laura Niederstaetter, Julia Brunmair, Martin Schaier, Benedikt Regner, Karin Nowikovsky, Christopher Gerner, Gunda Koellensperger, Petra Heffeter, Bernhard Klaus Keppler, Walter Berger. Lipid metabolism as target and modulator of KP1339 anticancer activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1839.