Karin Nowikovsky

Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy

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.

Muscle-Specific Loss of Apoptosis-Inducing Factor Leads to Mitochondrial Dysfunction, Skeletal Muscle Atrophy, and Dilated Cardiomyopathy

ABSTRACT 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.

Pathophysiology of mitochondrial volume homeostasis: Potassium transport and permeability transition

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.

The Pathophysiology of LETM1

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,

A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf-Hirschhorn syndrome

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.

Novel components of an active mitochondrial K(+)/H(+) exchange

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 mdm38Δ 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 ∼500-kDa protein complexes. Triple mutant strains (mdm38Δ mrs7Δ ydl183cΔ) exhibit a remarkably stronger pet− phenotype than mdm38Δ 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.

LETM1 in mitochondrial cation transport

LETM1 (leucine zipper- EF hand-containing transmembrane 1) encodes a highly conserved eukaryotic protein of the mitochondrial inner membrane which is essential to control mitochondrial volume homeostasis. Indeed, LETM1 RNA interference causes severe mitochondrial changes that include massive matrix swelling, loss of cristae structure and network fragmentation in S. cerevisiae (Nowikovsky et al., 2004), C. elegans (Hasegawa and van der Bliek, 2007), human cell cultures (Dimmer et al., 2008), D. melanogaster (McQuibban et al., 2010), and T. brucei (Hashimi et al., 2013). Conversely, its overexpression induces mitochondrial contraction and cristae condensation (Hasegawa and van der Bliek, 2007). LETM1 is associated with the Wolf-Hirschhorn Syndrome (WHS), a complex multigenic disease caused by haploinsufficiency of the WHSCR 1 and 2 regions on chromosome 4 (Endele et al., 1999). LETM1 is located less than 80 kb distal to WHSCR1 within the deleted locus in patients with seizures, and is preserved in all patients without seizures (Schlickum et al., 2004) implying LETM1 (hence mitochondrial dysfunction) in the pathogenesis of seizures. LETM1 has been originally proposed to be part of the mitochondrial K+-H+ exchanger (KHE) (Nowikovsky et al., 2004; Hasegawa and van der Bliek, 2007; Dimmer et al., 2008; McQuibban et al., 2010; Hashimi et al., 2013), an essential element of Mitchells chemiosmotic theory (Mitchell, 1966, 2011). The existence of a H+ electrochemical gradient driving electrophoretic K+ uptake indeed demands the existence of an electroneutral KHE to extrude K+ and thus, prevent osmotic burst of the organelle that would follow K+ accumulation (Mitchell, 1966, 2011). Strong support for a direct role of LETM1 in mitochondrial K+ homeostasis comes from the observation that inactivation of LETM1 (i) causes an increase of mitochondrial volume that can be reverted by nigericin (an ionophore that catalyzes the electroneutral exchange of H+ with K+) and (ii) can be phenocopied by the addition of valinomycin (an ionophore that catalyzes the electrophoretic flux of K+) (Nowikovsky et al., 2004; Froschauer et al., 2005; Dimmer et al., 2008; McQuibban et al., 2010; Hashimi et al., 2013). The idea that LETM1 catalyzes KHE (by itself or in combination with other proteins) was challenged by a genome-wide siRNA screening in Drosophila reporting that treatment with siRNA against LETM1 caused decreased Ca2+ influx into energized mitochondria; and that Ca2+ flux (proposed to be mediated by LETM1) was inhibited by ruthenium red (RR) (Jiang et al., 2009), the classical inhibitor of the mitochondrial Ca2+ uniporter, MCU (Moore, 1971). Based on these studies, it was suggested that LETM1 is a H+-Ca2+ antiporter catalyzing Ca2+ uptake in energized mitochondria (Jiang et al., 2009). As we have discussed in detail elsewhere, a H+-Ca2+ antiporter can catalyze Ca2+ uptake only if the H+/Ca2+ stoichiometry is lower than 2 (i.e., if net charge translocation takes place) while an antiporter with a stoichiometry of 2H+/Ca2+ can only catalyze Ca2+ efflux in energized mitochondria (Nowikovsky et al., 2012). A recent study bears on each of the questions outlined above (Tsai et al., 2014). LETM1-mediated Ca2+ transport was assessed by applying an inward Ca2+ gradient across liposomes containing highly purified recombinant LETM1, and then measuring Ca2+ flux either with a fluorescent indicator that had been trapped inside the liposomes, or with tracer 45Ca2+accumulation (Tsai et al., 2014). Using these approaches, LETM1-dependent Ca2+ flux was observed when a minimum of 30 μM Ca2+ was added outside the liposomes. Ca2+ flux was insensitive to valinomycin, suggesting that it was not affected by the membrane potential and therefore electroneutral; it was stimulated by a pH gradient, while other monovalent cations were ineffective suggesting that Ca2+ flux occurs in exchange for H+; and it was insensitive to RR (Tsai et al., 2014). These findings demonstrate that the RR-sensitive Ca2+ uptake reported previously (Jiang et al., 2009) could not be mediated by LETM1, and suggest that impaired Ca2+ uptake after LETM1 knock down was an indirect consequence of mitochondrial dysfunction and deenergization, an issue on which we will return later. The study of Tsai et al. also addressed the question of whether K+ is transported. LETM1-containing liposomes were loaded with K+ to obtain a 1000-fold outward K+ gradient, and then exposed to a trace amount of 86Rb+ which was not accumulated. The assumption was that an electroneutral KHE should also exchange K+ for K+ (Tsai et al., 2014), yet under the conditions of the experiment 86Rb+ distribution must follow that of K+, and the cation will be accumulated only if transport is electrophoretic. In other words, we believe that under the conditions of Tsai et al. LETM1 cannot catalyze KHE, and that to assess its occurrence a pH gradient should be imposed to the liposomes. The fact that both valinomycin and nigericin allowed 86Rb+ uptake into the liposomes (Tsai et al., 2014) adds to the problem, because with a 1000-fold outward K+ gradient 86Rb+ accumulation should be seen with valinomycin (which catalyzes electrophoretic K+/Rb+ transport) but not with nigericin which catalyzes strictly electroneutral K+/Rb+-H+ exchange, and would therefore require a pH gradient to catalyze 86Rb+ accumulation. Thus, we think that the study of Tsai et al. has not provided an answer as to whether LETM1 mediates KHE in a reconstituted system. There is no question that LETM1 inactivation causes severe mitochondrial dysfunction with depolarization and matrix swelling (Nowikovsky et al., 2004; Hasegawa and van der Bliek, 2007; Dimmer et al., 2008; McQuibban et al., 2010; Hashimi et al., 2013). Matrix swelling can be easily explained if LETM1 catalyzes KHE, because its inactivation would cause excessive matrix K+ uptake; but how could mitochondrial dysfunction and swelling be caused by inactivation of a 2H+-Ca2+ exchanger mediating Ca2+ efflux? Mitochondrial Ca2+ distribution is governed by the balance of electrophoretic Ca2+ uptake through the MCU (Baughman et al., 2011; De Stefani et al., 2011) and by Ca2+ release through the 3Na+-Ca2+ exchanger NCLX (Palty et al., 2010) and through a still unidentified Na+-insensitive Ca2+ release system, possibly a H+-Ca2+ antiporter, which is insensitive to RR (reviewed in Nowikovsky et al., 2012). In principle, inactivation of Ca2+ release in mammalian mitochondria could lead to Ca2+ overload and possibly to an increased open probability of the permeability transition pore (PTP), a high-conductance channel which forms from dimers of the F-ATP synthase (Giorgio et al., 2013) and mediates depolarization and mitochondrial swelling [reviewed in Bernardi (2013)]. Yet, operation of the ubiquitous NCLX should easily compensate for the absence of H+-Ca2+ exchange. Thus, at present there is a solid mechanism to explain mitochondrial dysfunction following LETM1 inactivation only if LETM1 takes part in KHE. The LETM1 gene has been evolutionarily conserved from yeast to trypanosomes, to Drosophila and mammals (Nowikovsky et al., 2004; Hasegawa and van der Bliek, 2007; Dimmer et al., 2008; McQuibban et al., 2010; Hashimi et al., 2013), suggesting that is serves a common function in these organisms. Yeast mitochondria do not possess an MCU, and therefore they cannot catalyze rapid Ca2+ uptake (Carafoli and Lehninger, 1971). It appears thus difficult to envision why yeast mitochondria should have developed a Ca2+ release mechanism which can only lead to matrix Ca2+ depletion. Since yeast mitochondria undergo in situ swelling when LETM1 is inactivated -an event that cannot be ascribed to Ca2+ overload- we still favor the hypothesis that LETM1 catalyzes KHE in situ, as overwhelming evidence suggests (Nowikovsky et al., 2004; Hasegawa and van der Bliek, 2007; Dimmer et al., 2008; McQuibban et al., 2010; Hashimi et al., 2013). Can these apparently conflicting sets of data be reconciled? It is possible that cation selectivity in situ is affected by protein interactions that are lost in the reconstituted system, and thus that LETM1 catalyzes RR-insensitive, electroneutral exchange of H+ with both K+ and Ca2+, an issue that should be addressed in future studies of this fascinating problem.

Calpain-Mediated Integrin Deregulation as a Novel Mode of Action for the Anticancer Gallium Compound KP46

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 Mg2+ 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. Mol Cancer Ther; 13(10); 2436–49. ©2014 AACR.

Unique membrane-interacting properties of the immunostimulatory cationic peptide KLKL5KLK (KLK)

We have monitored the effects of KLKL5KLK (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 KLKs 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™.

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells

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.