Mitochondrial Targeting Sequence Research Articles

Clinical, Molecular, and Computational Analysis in two cases with mitochondrial encephalomyopathy associated with SUCLG1 mutation in a consanguineous family

Deficiency of the mitochondrial enzyme succinyl COA ligase (SUCL) is associated with encephalomyopathic mtDNA depletion syndrome and methylmalonic aciduria. This disorder is caused by mutations in both SUCL subunits genes: SUCLG1 (α subnit) and SUCLA2 (β subnit). We report here, two Tunisian patients belonging to a consanguineous family with mitochondrial encephalomyopathy, hearing loss, lactic acidosis, hypotonia, psychomotor retardation and methylmalonic aciduria. Mutational analysis of SUCLG1 gene showed, for the first time, the presence of c.41T > C in the exon 1 at homozygous state. In-silico analysis revealed that this mutation substitutes a conserved methionine residue to a threonine at position 14 (p.M14T) located at the SUCLG1 protein mitochondrial targeting sequence. Moreover, these analysis predicted that this mutation alter stability structure and mitochondrial translocation of the protein. In Addition, a decrease in mtDNA copy number was revealed by real time PCR in the peripheral blood leukocytes in the two patients compared with controls.

A conserved mammalian mitochondrial isoform of acetyl-CoA carboxylase ACC1 provides the malonyl-CoA essential for mitochondrial biogenesis in tandem with ACSF3.

Mitochondrial fatty acid synthesis (mtFAS) is a highly conserved pathway essential for mitochondrial biogenesis. The mtFAS process is required for mitochondrial respiratory chain assembly and function, synthesis of the lipoic acid cofactor indispensable for the function of several mitochondrial enzyme complexes and essential for embryonic development in mice. Mutations in human mtFAS have been reported to lead to neurodegenerative disease. The source of malonyl-CoA for mtFAS in mammals has remained unclear. We report the identification of a conserved vertebrate mitochondrial isoform of ACC1 expressed from an ACACA transcript splicing variant. A specific knockdown (KD) of the corresponding transcript in mouse cells, or CRISPR/Cas9-mediated inactivation of the putative mitochondrial targeting sequence in human cells, leads to decreased lipoylation and mitochondrial fragmentation. Simultaneous KD of ACSF3, encoding a mitochondrial malonyl-CoA synthetase previously implicated in the mtFAS process, resulted in almost complete ablation of protein lipoylation, indicating that these enzymes have a redundant function in mtFAS. The discovery of a mitochondrial isoform of ACC1 required for lipoic acid synthesis has intriguing consequences for our understanding of mitochondrial disorders, metabolic regulation of mitochondrial biogenesis and cancer.

Comparison on the Roles of COQ3 and COQ7 Proteins in Maintaining Coenzyme Q 10 Levels, the Stability of Other COQ Proteins, and Mitochondrial Functions in Human Cells

Coenzyme Q (CoQ) is a lipid-soluble antioxidant, with the oxidized (ubiquinone) and the reduced (ubiquinol) forms, and also an electron carrier for the electron transport chain. CoQ6 is synthesized by at least nine proteins (Coq1--9), and many of them form the Coq protein complex in the mitochondria in yeast. CoQ is present in the form of CoQ10 in humans. Yeast Coq1 corresponds to PDSS1 and PDSS2 proteins in humans. Mutations in PDSS or COQ genes are associated with primary CoQ10 deficiency. Secondary CoQ10 deficiency is associated with mitochondrial DNA mutation. We have demonstrated that the human COQ5, present as a mature protein without mitochondrial targeting sequence in the mitochondria, is required for maintaining CoQ10 levels. The maturation of COQ5 protein and the stability of the COQ5-containing protein complex in the mitochondria were suppressed by the conditions of mitochondrial energy deficiency. Here we further investigated the roles of COQ3 and COQ7 proteins in maintaining CoQ10 levels and in the mitochondria. Our results showed that both COQ3 and COQ7 were required for maintaining CoQ10 levels in human 143B cells, but the degree of the decrease in CoQ10 levels was greater after COQ7 knockdown than that after COQ3 knockdown. After COQ7 knockdown, but not after COQ3 knockdown, the ubiquinol-10:ubiquinone-10 ratio was increased and the peaks corresponding to the putative substrate of the COQ7 enzyme, 5-demethoxyubiquinone-10 (DMQ10/DMQ10H2), appeared on the HPLC chromatograms. The CoQ10 levels in the mitochondria were also decreased after COQ3 or COQ7 knockdown, and the putative DMQ10 was present in the mitochondria after COQ7 knockdown. Moreover, COQ3 or COQ7 knockdown caused decreased mitochondrial respiratory functions. Only COQ3 knockdown could decrease the stability of PDSS2 and several COQ proteins. Furthermore, mature forms of COQ3 and COQ7 were found in the mitochondria. Levels of the mature COQ7 protein, but not COQ3, were suppressed in 143B cells treated with the chemical uncoupler FCCP and in the cybrids harboring a pathogenic mitochondrial DNA mutation, which both had diminished CoQ10 levels. All these novel findings may further elucidate the functions of COQ proteins and their roles in human diseases.

Abstract 3890: Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance

Abstract Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. MCL1 is an anti-apoptotic Bcl-2 family member known to sequester and inactivate pro-apoptotic Bcl-2 family proteins and, thus, contribute to chemotherapy resistance. We previously reported that ~45% of residual TNBCs that remain in the breast after neoadjuvant chemotherapy harbor MCL1 amplification, suggesting a causal role for MCL1 in drug resistance. A recent report (Goodwin et al. 2015) suggested that siRNA-mediated ablation of MCL1 does not induce apoptosis in claudin-low TNBC cells with a cancer stem cell (CSC) gene expression signature. CSCs comprise a rare population of cells with tumor-initiating properties and refractoriness to chemotherapy. In this study, we showed that MCL1 expression is elevated in claudin-low TNBC SUM159PT and MDA436 CSCs as measured by ALDH+ by flow cytometry and ability to form mammospheres. RNA interference of MCL1 in SUM159PT cells reduced CSCs and attenuated tumor formation in vivo. Mitochondrial oxidative phosphorylation (mtOXPHOS) plays a crucial role in maintenance of CSCs. MCL1 has been shown to localize in the mitochondrial matrix and contribute to mitochondrial respiration. Thus, we hypothesized that MCL1 contributes to enrichment of TNBC CSCs and chemotherapy resistance via mitochondrial regulation. Stable transfection and overexpression of MCL1 in MDA468 cells increased oxygen consumption ratio, mitochondrial membrane potential, and production of reactive oxygen species (ROS), all features of activated mtOXPHOS. Conversely, RNAi-mediated ablation of MCL1 in SUM159PT and MDA436 cells repressed these markers of activated mtOXPHOS. A mutant of MCL1 lacking its mitochondrial target sequences (MTS) was unable to localize in mitochondria and, when transfected into MDA468 cells, reduced the CD44high/CD24low fraction and mammosphere formation. We next tested VU0659158, a BH3 mimetic in development at Vanderbilt that disrupts MCL1 interactions with BH3 domain-containing proteins, such as BID, BIM, NOXA and PUMA. Treatment of SUM159PT cells with VU0659158 increased caspase activity but did not attenuate mammosphere formation. Analysis of mRNA expression in TCGA revealed that genes induced by mtOXPHOS involved in the hypoxia pathway are significantly up-regulated in MCL1 amplified breast cancers. Finally, pharmacological inhibition of HIF-1α, a key regulator of hypoxia, with digoxin decreased CSCs and attenuated tumor formation in vivo. These data suggest that 1) MCL1 confers resistance to chemotherapy by expanding CSCs via mtOXPHOS independent of its BH3 domain-mediated, anti-apoptotic function, and 2) targeting mitochondrial respiration and the hypoxia pathway may delay or reverse chemotherapy resistance in MCL1 amplified TNBC. Citation Format: Kyung-min Lee, Jennifer Giltnane, Justin Balko, Luis Schwarz, Angel Guerrero, Katie Hutchinson, Mellissa Hicks, Violeta Sanchez, Melinda Sanders, Taekyu Lee, Edward Olejniczak, Stephen Fesik, Carlos Arteaga. Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2017-3890

Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death.

Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis.

The molecular characterizations of Cu/ZnSOD and MnSOD and its responses of mRNA expression and enzyme activity to Aeromonas hydrophila or lipopolysaccharide challenge in Qihe crucian carp Carassius auratus

Superoxide dismutases (SODs), as the prime antioxidant enzymes, present the first line of defense against oxidative stress caused by excessive reactive oxygen species (ROS) in organism. In the study, two distinct members of SOD family were cloned and analyzed in Qihe crucian carp Carassius auratus (designated as CaCu/ZnSOD and CaMnSOD, respectively). The full-length cDNA of CaCu/ZnSOD is 759 bp, containing a 5' -untranslated region (UTR) of 39 bp, a ORF (including stop codon, TAG) of 465 bp and a 3′-UTR of 255 bp. The ORF of CaCu/ZnSOD encodes a protein of 154 amino acids (aa), in which, two Cu/ZnSOD signature (45GFHVHAFGDNT55 and 139GNAGGRLACGVI150) and four conserved amino acids for Cu/Zn2+-binding sites (H64, H72, H81 and D84) were observed. The full-length CaMnSOD cDNA (960 bp) consists of a 5′-UTR of 114 bp, a ORF of 675 bp and a 3′-UTR of 231 bp, the ORF of CaMnSOD encodes a 224 aa protein with a 26 aa mitochondrial-targeting sequence (MTS) in the N-terminus, and four conserved amino acids for manganese binding (H52, H100, D185 and H189) were observed. Multiple alignment and the structural analysis revealed two Cu/ZnSOD signature motifs and a MnSOD signature motif as well as the invariant binding sites for Cu2+/Zn2+ in CaCu/ZnSOD and Mn2+ in CaMnSOD. The phylogenetic analysis indicated that CaCu/ZnSOD was homologous to cytosolic Cu/ZnSODs, and CaMnSOD was high similarity with mitochondrial MnSODs from other fish. The tissue distribution analysis demonstrated that CaCu/ZnSOD and CaMnSOD were highly expressed in liver, heart and muscle. The dynamic expressions of CaCu/ZnSOD and CaMnSOD were observed after the challenges with Aeromonas hydrophila or LPS, which generally increased in liver, gill, kidney and spleen, while, the mRNA expressions were down-regulated at some time points in head kidney. The enzyme activities increased after A. hydrophila or LPS challenge, compared to the control. In this study, the molecular structures and functional motifs of CaCu/ZnSOD and CaMnSOD were determined, and it is crucial for us to understand the biological functions of SODs. The highest level in liver showed that the function of liver to remove ROS is much more important. The obvious responses of mRNA expression levels and enzyme activities to pathogens indicate the important roles of CaCu/ZnSOD and CaMnSOD in antioxidant defense in C. auratus.

Is detection of intraperitoneal exfoliated tumor cells after surgical resection of rectal cancer a prognostic factor of survival?

BackgroundThe prognostic significance of free cancer cells detected in peritoneal fluid at the time of rectal surgery remains unclear. A substantial number of patients will develop metastatic disease even with successful local treatment. This prospective non-randomized study investigated the prognostic value of intraperitoneal free cancer cells harvested in peritoneal lavage after surgery for rectal cancer. Mutational hotspots in mitochondrial DNA were examined as potential molecular signatures to detect circulating intraperitoneal free cancer cells when present in primary tumor and in lavage.MethodsPoint mutations in mitochondrial DNA amplifications were determined in primary tumors and corresponding exfoliated intraperitoneal free cancer cells in lavage from 191 patients with locally advanced rectal cancer scheduled for radical treatment. Mitochondrial DNA target sequences were amplified by polymerase chain reaction and base substitutions were detected by denaturant, cycling temperature capillary electrophoresis. Detection of intraperitoneal free cancer cells was correlated to survival.ResultsOf 191patients analyzed, 138 (72%) were identified with somatic mitochondrial point mutations in rectal cancer tumors. From this fraction, 45 patients (33%) had positive lavage fluid with corresponding somatic mtDNA point mutations in lavage representing circulating intraperitoneal free cancer cells. There was no significant survival difference between patients identified with or without somatic mitochondrial DNA point mutations in the corresponding lavage.ConclusionSomatic mitochondrial DNA point mutations identified in primary rectal tumors enable detection of circulating intraperitoneal free cancer cells in lavage fluid.Intraperitoneal free cancer cells harvested from lavage immediately after surgery for rectal cancer does not represent an independent prognostic factor on survival.

Characterization of the novel protein KIAA0564 (Von Willebrand Domain-containing Protein 8)

The VWA8 gene was first identified by the Kazusa cDNA project and named KIAA0564. Based on the observation, by similarity, that the protein encoded by KIAA0564 contains a Von Willebrand Factor 8 domain, KIAA0564 was named Von Willebrand Domain-containing Protein 8 (VWA8). The function of VWA8 protein is almost unknown. The purpose of this study was to characterize the tissue distribution, cellular location, and function of VWA8. In mice VWA8 protein was mostly distributed in liver, kidney, heart, pancreas and skeletal muscle, and is present as a long isoform and a shorter splice variant (VWA8a and VWA8b). VWA8 protein and mRNA were elevated in mouse liver in response to high fat feeding. Sequence analysis suggests that VWA8 has a mitochondrial targeting sequence and domains responsible for ATPase activity. VWA8 protein was targeted exclusively to mitochondria in mouse AML12 liver cells, and this was prevented by deletion of the targeting sequence. Moreover, the VWA8 short isoform overexpressed in insect cells using a baculovirus construct had in vitro ATPase activity. Deletion of the Walker A motif or Walker B motif in VWA8 mostly blocked ATPase activity, suggesting Walker A motif or Walker B motif are essential to the ATPase activity of VWA8. Finally, homology modeling suggested that VWA8 may have a structure most confidently similar to dynein motor proteins.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.

We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane β-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM- and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22- and mtHsp70-dependent import pathway.

VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System.

VopE, a mitochondrial targeting T3SS effector protein of Vibrio cholerae, perturbs innate immunity by modulating mitochondrial dynamics. In the current study, ectopic expression of VopE was found to be toxic in a yeast model system and toxicity was further aggravated in the presence of various stressors. Interestingly, a VopE variant lacking predicted mitochondrial targeting sequence (MTS) also exhibited partial lethality in the yeast system. With the aid of yeast genetic tools and different stressors, we have demonstrated that VopE and its derivative VopEΔMTS modulate cell wall integrity (CWI-MAPK) signaling pathway and have identified several critical residues contributing to the lethality of VopE. Furthermore, co-expression of two effectors VopEΔMTS and VopX, interfering with the CWI-MAPK cellular pathway can partially suppress the VopX mediated yeast growth inhibition. Taken together, these results suggest that VopE alters signaling through the CWI-MAPK pathway, and demonstrates the usefulness of yeast model system to gain additional insights on the functionality of VopE.

The N-Terminus of Iron-Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with l-Cysteine Desulfurase NFS1.

Assembly of iron-sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by l-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondrial targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved "LYR" motif is essential for the role of ISD11 in forming a stable and active complex with NFS1.

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

Evolutionary origin of mitochondrial cytochrome P450.

Different molecular species of cytochrome P450 (P450) are distributed between endoplasmic reticulum (microsomes) and mitochondria in animal cells. Plants and fungi have many microsomal P450s, but no mitochondrial P450 has so far been reported. To elucidate the evolutionary origin of mitochondrial P450s in animal cells, available evidence is examined, and the virtual absence of mitochondrial P450 in plants and fungi is confirmed. It is also suggested that a microsomal P450 is the ancestor of animal mitochondrial P450s. It is likely that the endoplasmic reticulum-targeting sequence at the amino-terminus of a microsomal P450 was converted to a mitochondria-targeting sequence possibly by point mutations of a few amino acid residues or by an exon-shuffling/moving event shortly after animal lineage diverged from plants and fungi in the course of evolution of eukaryotes. It is suggested that the microsome-type P450 first imported into mitochondria utilized the existing ferredoxin in the matrix to receive electrons from NADPH, retained its oxygenase activity in the mitochondria, and gradually diversified to several P450s with different substrate specificities in the course of the evolution of animals.

An Alternative Splice Variant of Chloride Intracellular Channel 5 Protein, (CLIC5B) Regulates Cardiac Mitochondrial Localization and Function of CLIC5

Mitochondrial bioenergetics is critical for the cell survival. Mitochondrial ion channels like BKCa and KATP play a significant role in cardio-protection from ischemia-reperfusion (IR) injury possibly by modulating the mitochondrial physiology. Interestingly, inner mitochondrial membrane (IMM) channels lack canonical mitochondrial targeting sequence (MTS) but localize to mitochondria. In case of mitoBKCa, a c-terminal splice variant, DEC, (BKCa-DEC) was reported to target BKCa to mitochondria of adult cardiomyocytes.

Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death.

Although the second messenger cyclic AMP (cAMP) is physiologically beneficial in the heart, it largely contributes to cardiac disease progression when dysregulated. Current evidence suggests that cAMP is produced within mitochondria. However, mitochondrial cAMP signaling and its involvement in cardiac pathophysiology are far from being understood. To investigate the role of MitEpac1 (mitochondrial exchange protein directly activated by cAMP 1) in ischemia/reperfusion injury. We show that Epac1 (exchange protein directly activated by cAMP 1) genetic ablation (Epac1-/-) protects against experimental myocardial ischemia/reperfusion injury with reduced infarct size and cardiomyocyte apoptosis. As observed in vivo, Epac1 inhibition prevents hypoxia/reoxygenation-induced adult cardiomyocyte apoptosis. Interestingly, a deleted form of Epac1 in its mitochondrial-targeting sequence protects against hypoxia/reoxygenation-induced cell death. Mechanistically, Epac1 favors Ca2+ exchange between the endoplasmic reticulum and the mitochondrion, by increasing interaction with a macromolecular complex composed of the VDAC1 (voltage-dependent anion channel 1), the GRP75 (chaperone glucose-regulated protein 75), and the IP3R1 (inositol-1,4,5-triphosphate receptor 1), leading to mitochondrial Ca2+ overload and opening of the mitochondrial permeability transition pore. In addition, our findings demonstrate that MitEpac1 inhibits isocitrate dehydrogenase 2 via the mitochondrial recruitment of CaMKII (Ca2+/calmodulin-dependent protein kinase II), which decreases nicotinamide adenine dinucleotide phosphate hydrogen synthesis, thereby, reducing the antioxidant capabilities of the cardiomyocyte. Our results reveal the existence, within mitochondria, of different cAMP-Epac1 microdomains that control myocardial cell death. In addition, our findings suggest Epac1 as a promising target for the treatment of ischemia-induced myocardial damage.

Inhibition of mitochondrial calcium uptake 1 in Drosophila neurons.

The mitochondrial calcium uptake 1 (MICU1) is a regulatory subunit of the mitochondrial calcium uniporter that plays an important role in calcium sensing. It contains two EF-hand domains that are well conserved across diverse species from protozoa to plants and metazoans. The loss of MICU1 function in mammals is attributed to several neurological disorders that involve movement dysfunction. The CG4495 gene in Drosophila melanogaster was identified as a putative homolog of MICU1 in the HomoloGene database of the National Centre for Biotechnology Information (NCBI). In agreement with previous studies that have shown the development of neurological disorders and movement defects in MICU1 loss-of-function organisms, we attempted to identify the function of CG4495/MICU1 in Drosophila neurons. We analyzed survival and locomotor ability of these flies and additionally performed biometric analysis of the Drosophila developing eye. The inducible RNA interference-mediated inhibition of CG4495/MICU1 in the Ddc-Gal4-expressing neurons of Drosophila presented with reduction in survival coupled with a precocious loss of locomotor ability. Since the pro-survival Bcl-2 family genes have been shown to be protective towards mitochondria, and CG4495/MICU1 has a mitochondrial targeting sequence, we attempted to rescue the phenotypes resulting from the inhibition of CG4495/MICU1 by overexpressing Buffy, the sole Bcl-2 homologue in Drosophila. The co-expression of CG4495/MICU1-RNAi along with Buffy resulted in the suppression of the phenotypes induced by the inhibition of CG4495/MICU1. Subsequently, the inhibition of CG4495/MICU1 in the Drosophila developing eye, a neuron-rich organ, resulted in reduced number of ommatidia and a highly fused ommatidial array. These developmental eye defects were rescued by the overexpression of Buffy. Our study suggests an important role for MICU1 in the normal function of neurons in Drosophila.

Anatomical distributional defects in mutant genes associated with dominant intermediate Charcot-Marie-Tooth disease type C in an adenovirus-mediated mouse model.

Dominant intermediate Charcot-Marie-Tooth disease type C (DI-CMTC) is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features. DI-CMTC is linked to tyrosyl-tRNA synthetase (YARS)-associated neuropathies, which are caused by E196K and G41R missense mutations and a single de novo deletion (153-156delVKQV). It is well-established that these YARS mutations induce neuronal dysfunction, morphological symptoms involving axonal degeneration, and impaired motor performance. The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria. An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord, peripheral axons, and dorsal root ganglia. Following transfection of YARS-expressing viruses, the distributions of wild-type (WT) YARS and E196K mutant proteins were compared in all expressed regions; G41R was not expressed. The proportion of Flag/green fluorescent protein (GFP) double-positive signaling in the E196K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons. All adenovirus genes, and even the empty vector without the YARS gene, exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter. The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes. Thus, use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons, the DI-CMTC phenotype, and potential treatment approaches for this disease.

438 - Genetically Encoded Tools for Compartment-Specific Manipulation of NAD+/NADH or NADP+/NADPH Ratios

Background The pool sizes and ratios of the pyridine nucleotides NAD(P)+ and NAD(P)H play a fundamental role in cellular metabolism and have emerged as key factors in numerous pathologies, including mitochondrial disorders, neurodegenerative diseases, cancer and aging. However, evaluating the impact of changes in concentrations of particular pyridine nucleotides in these conditions has not been possible due to lack of tools with which to directly perturb the NAD(P)+/NAD(P)H ratios. The aim of our study was to develop such tools, which are based on heterologous expression of naturally occurring or engineered water-forming NAD(P)H oxidases (NOXes). NOXes catalyze a four-electron reduction of oxygen to water using reducing equivalents of NAD(P)H, and their natural function is to detoxify O2 in several facultative or strictly anaerobic bacteria and parasitic protozoa. Methods Naturally occurring eukaryotic and bacterial NOXes were screened in human cells with or without a mitochondrial targeting sequence and assessed for their ability to consume oxygen and to change NAD(P)+/NAD(P)H ratios. The best candidates were overproduced in E.coli, purified and biochemically characterized for their substrate specificity and activity. Structural enzymology and rational mutagenesis were employed to engineer NAD(P)H specific variants. Results Our kinetic and structural studies revealed that bacterial NOX from Lactobacillus brevis (LbNOX) is strictly specific for NADH over NADPH, produces negligible H2O2, and has high catalytic activity compared to other candidate NOXes. Expression of LbNOX in mitochondria or cytoplasm of human cells is well tolerated and leads to a compartment-specific increase in the NAD+/NADH ratio. We used LbNOX to demonstrate the dependence of key metabolic fluxes and signaling on the cytosolic or mitochondrial NAD+/NADH ratios. In addition, LbNOX can fully complement electron transport chain function to support mammalian cell proliferation by re-oxidizing NADH to NAD+. Conclusions Taken together, the genetically encoded tools which we are developing provide a toolkit for compartment-specific manipulation of the NAD+/NADH and NADP+/NADPH ratios in living cells.

Overlapping Role of Respiratory Supercomplex Factor Rcf2 and Its N-terminal Homolog Rcf3 in Saccharomyces cerevisiae

The mitochondrial electron transport chain consists of individual protein complexes arranged into large macromolecular structures, termed respiratory chain supercomplexes or respirasomes. In the yeast Saccharomyces cerevisiae, respiratory chain supercomplexes form by association of the bc1 complex with the cytochrome c oxidase. Formation and maintenance of these assemblies are promoted by specific respiratory supercomplex factors, the Rcf proteins. For these proteins a regulatory function in bridging the electron transfer within supercomplexes has been proposed. Here we report on the maturation of Rcf2 into an N- and C-terminal peptide. We show that the previously uncharacterized Rcf3 (YBR255c-A) is a homolog of the N-terminal Rcf2 peptide, whereas Rcf1 is homologous to the C-terminal portion. Both Rcf3 and the C-terminal fragment of Rcf2 associate with monomeric cytochrome c oxidase and respiratory chain supercomplexes. A lack of Rcf2 and Rcf3 increases oxygen flux through the respiratory chain by up-regulation of the cytochrome c oxidase activity. A double gene deletion of RCF2 and RCF3 affects cellular survival under non-fermentable growth conditions, suggesting an overlapping role for both proteins in the regulation of the OXPHOS activity. Furthermore, our data suggest an association of all three Rcf proteins with the bc1 complex in the absence of a functional cytochrome c oxidase and identify a supercomplex independent interaction network of the Rcf proteins.

Getting into mitochondria.

The human mitochondrial glutamate dehydrogenase isoenzymes (hGDH1 and hGDH2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predictions suggest the presence of two α-helices within the N-terminal region of the MTS. Results from deletion analyses indicate that individual helices have limited ability to direct protein import and matrix localization, but that there is a synergistic interaction when both helices are present [Biochem. J. (2016) 473: , 2813-2829]. Mutagenesis of the MTS cleavage sites blocked post-import removal of the presequences, but did not impede import. The authors propose that the high matrix levels of hGDH can be attributed to the unusual length and secondary structure of the MTS.