Mitochondrial Aminoacyl-tRNA Synthetase and Disease: The Yeast Contribution for Functional Analysis of Novel Variants

In most eukaryotes, mitochondrial protein synthesis is essential for oxidative phosphorylation (OXPHOS) as some subunits of the respiratory chain complexes are encoded by the mitochondrial DNA (mtDNA). Mutations affecting the mitochondrial translation apparatus have been identified as a major cause of mitochondrial diseases. These mutations include either heteroplasmic mtDNA mutations in genes encoding for the mitochondrial rRNA (mtrRNA) and tRNAs (mttRNAs) or mutations in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases (mtARSs). Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs. Differently from most mttRNAs, which are encoded by mitochondrial genome, mtARSs are encoded by nuclear genes and then imported into the mitochondria after translation in the cytosol. Due to the extensive use of next-generation sequencing (NGS), an increasing number of mtARSs variants associated with large clinical heterogeneity have been identified in recent years. Being most of these variants private or sporadic, it is crucial to assess their causative role in the disease by functional analysis in model systems. This review will focus on the contributions of the yeast Saccharomyces cerevisiae in the functional validation of mutations found in mtARSs genes associated with human disorders.     

A Moonlighting Human Protein Is Involved in Mitochondrial Import of tRNA

In yeast Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2).     

Diverse Functions of Tim50, a Component of the Mitochondrial Inner Membrane Protein Translocase

Mitochondria are essential in eukaryotes. Besides producing 80% of total cellular ATP, mitochondria are involved in various cellular functions such as apoptosis, inflammation, innate immunity, stress tolerance, and Ca²⁺ homeostasis. Mitochondria are also the site for many critical metabolic pathways and are integrated into the signaling network to maintain cellular homeostasis under stress. Mitochondria require hundreds of proteins to perform all these functions. Since the mitochondrial genome only encodes a handful of proteins, most mitochondrial proteins are imported from the cytosol via receptor/translocase complexes on the mitochondrial outer and inner membranes known as TOMs and TIMs. Many of the subunits of these protein complexes are essential for cell survival in model yeast and other unicellular eukaryotes. Defects in the mitochondrial import machineries are also associated with various metabolic, developmental, and neurodegenerative disorders in multicellular organisms. In addition to their canonical functions, these protein translocases also help maintain mitochondrial structure and dynamics, lipid metabolism, and stress response. This review focuses on the role of Tim50, the receptor component of one of the TIM complexes, in different cellular functions, with an emphasis on the Tim50 homologue in parasitic protozoan Trypanosoma brucei.     

Proteome Analysis of Subsarcolemmal Cardiomyocyte Mitochondria: A Comparison of Different Analytical Platforms

Mitochondria are complex organelles that play critical roles in diverse aspects of cellular function. Heart disease and a number of other pathologies are associated with perturbations in the molecular machinery of the mitochondria. Therefore, comprehensive, unbiased examination of the mitochondrial proteome represents a powerful approach toward system-level insights into disease mechanisms. A crucial aspect in proteomics studies is design of bioanalytical strategies that maximize coverage of the complex repertoire of mitochondrial proteins. In this study, we evaluated the performance of gel-based and gel-free multidimensional platforms for profiling of the proteome in subsarcolemmal mitochondria harvested from rat heart. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and bioinformatics for protein identification. Across all three platforms, a total of 1043 proteins were identified. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage of the mitochondrial proteome. With this platform, 890 proteins with diverse physicochemical characteristics were identified; the mitochondrial protein panel encompassed proteins with various functional roles including bioenergetics, protein import, and mitochondrial fusion. Taken together, results of this study provide a large-scale view of the proteome in subsarcolemmal mitochondria from the rat heart, and aid in the selection of optimal bioanalytical platforms for differential protein expression profiling of mitochondria in health and disease.     

Identification of Phosphorylated Calpain 3 in Rat Brain Mitochondria under mPTP Opening

The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60–62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca²⁺ efflux from mitochondria, preventing the opening of mPTP. It was found that phosphorylated truncated CALP3 with a molecular weight of 60–62 contains p-Tyr, which indicates the possible involvement of protein tyrosine phosphatase in this process.     

The Antibiotic Doxycycline Impairs Cardiac Mitochondrial and Contractile Function

Tetracycline antibiotics act by inhibiting bacterial protein translation. Given the bacterial ancestry of mitochondria, we tested the hypothesis that doxycycline—which belongs to the tetracycline class—reduces mitochondrial function, and results in cardiac contractile dysfunction in cultured H9C2 cardiomyoblasts, adult rat cardiomyocytes, in Drosophila and in mice. Ampicillin and carbenicillin were used as control antibiotics since these do not interfere with mitochondrial translation. In line with its specific inhibitory effect on mitochondrial translation, doxycycline caused a mitonuclear protein imbalance in doxycycline-treated H9C2 cells, reduced maximal mitochondrial respiration, particularly with complex I substrates, and mitochondria appeared fragmented. Flux measurements using stable isotope tracers showed a shift away from OXPHOS towards glycolysis after doxycycline exposure. Cardiac contractility measurements in adult cardiomyocytes and Drosophila melanogaster hearts showed an increased diastolic calcium concentration, and a higher arrhythmicity index. Systolic and diastolic dysfunction were observed after exposure to doxycycline. Mice treated with doxycycline showed mitochondrial complex I dysfunction, reduced OXPHOS capacity and impaired diastolic function. Doxycycline exacerbated diastolic dysfunction and reduced ejection fraction in a diabetes mouse model vulnerable for metabolic derangements. We therefore conclude that doxycycline impairs mitochondrial function and causes cardiac dysfunction.     

Overlapping Functions of the Paralogous Proteins AtPAP2 and AtPAP9 in Arabidopsis thaliana

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), which is anchored to the outer membranes of chloroplasts and mitochondria, affects carbon metabolism by modulating the import of some preproteins into chloroplasts and mitochondria. AtPAP9 bears a 72% amino acid sequence identity with AtPAP2, and both proteins carry a hydrophobic motif at their C-termini. Here, we show that AtPAP9 is a tail-anchored protein targeted to the outer membrane of chloroplasts. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that both AtPAP9 and AtPAP2 bind to a small subunit of rubisco 1B (AtSSU1B) and a number of chloroplast proteins. Chloroplast import assays using [³⁵S]-labeled AtSSU1B showed that like AtPAP2, AtPAP9 also plays a role in AtSSU1B import into chloroplasts. Based on these data, we propose that AtPAP9 and AtPAP2 perform overlapping roles in modulating the import of specific proteins into chloroplasts. Most plant genomes contain only one PAP-like sequence encoding a protein with a hydrophobic motif at the C-terminus. The presence of both AtPAP2 and AtPAP9 in the Arabidopsis genome may have arisen from genome duplication in Brassicaceae. Unlike AtPAP2 overexpression lines, the AtPAP9 overexpression lines did not exhibit early-bolting or high-seed-yield phenotypes. Their differential growth phenotypes could be due to the inability of AtPAP9 to be targeted to mitochondria, as the overexpression of AtPAP2 on mitochondria enhances the capacity of mitochondria to consume reducing equivalents.     

Effects of Liposome and Cardiolipin on Folding and Function of Mitochondrial Erv1

Erv1 (EC number 1.8.3.2) is an essential mitochondrial enzyme catalyzing protein import and oxidative folding in the mitochondrial intermembrane space. Erv1 has both oxidase and cytochrome c reductase activities. While both Erv1 and cytochrome c were reported to be membrane associated in mitochondria, it is unknown how the mitochondrial membrane environment may affect the function of Erv1. Here, in this study, we used liposomes to mimic the mitochondrial membrane and investigated the effect of liposomes and cardiolipin on the folding and function of yeast Erv1. Enzyme kinetics of both the oxidase and cytochrome c reductase activity of Erv1 were studied using oxygen consumption analysis and spectroscopic methods. Our results showed that the presence of liposomes has mild impacts on Erv1 oxidase activity, but significantly inhibited the catalytic efficiency of Erv1 cytochrome c reductase activity in a cardiolipin-dependent manner. Taken together, the results of this study provide important insights into the function of Erv1 in the mitochondria, suggesting that molecular oxygen is a better substrate than cytochrome c for Erv1 in the yeast mitochondria.     

Leucyl-tRNA Synthetase Regulates Lactation and Cell Proliferation via mTOR Signaling in Dairy Cow Mammary Epithelial Cells

The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.     

Studies on liver phosphatidyl cholines: I. Effects of fatty liver induction on phosphatidyl cholines from liver mitochondria and microsomes

Protein, total phospholipid, phosphatidyl cholines and phosphatidyl choline fractions from liver mitochondria and microsomes of female rats were analyzed after treatment with CCl₄ (0.3 ml of CCl₄ suspended in corn oil) or ethionine (50 mg in 0.9% saline) or after feeding a choline deficient, low protein diet for seven days. Phosphatidyl cholines were separated into four fractions differing in the degree of fatty acid unsaturation. Over 50% of total phosphatidyl choline phosphorus was present in fraction 3 of liver mitochondria and microsomes. The major fatty acid in fraction 1 was docosahexaenoic acid. Fraction 4 contains oleic and linoleic acids. Arachidonic acid occurs in fraction 2 and 3. Ethionine decreased the amount of microsomal protein and phosphatidyl choline fraction 1 of mitochondria. Microsomal protein was decreased by CCl₄. The choline deficient, low protein diet caused a decrease in mitochondrial and microsomal phospholipids. The amount of the mitochondrial phosphatidyl choline decreased. Corn oil increased the level of phosphatidyl choline fraction 3. Choline deficiency decreased the amount of phosphatidyl choline fraction 3, increased fraction 4 of mitochondria and microsomes and increased fraction 1 of microsomes.     

Folding and Biogenesis of Mitochondrial Small Tim Proteins

Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.     

Characterization of a Novel Splicing Variant in Acylglycerol Kinase (AGK) Associated with Fatal Sengers Syndrome

Mitochondrial functional integrity depends on protein and lipid homeostasis in the mitochondrial membranes and disturbances in their accumulation can cause disease. AGK, a mitochondrial acylglycerol kinase, is not only involved in lipid signaling but is also a component of the TIM22 complex in the inner mitochondrial membrane, which mediates the import of a subset of membrane proteins. AGK mutations can alter both phospholipid metabolism and mitochondrial protein biogenesis, contributing to the pathogenesis of Sengers syndrome. We describe the case of an infant carrying a novel homozygous AGK variant, c.518+1G>A, who was born with congenital cataracts, pielic ectasia, critical congenital dilated myocardiopathy, and hyperlactacidemia and died 20 h after birth. Using the patient’s DNA, we performed targeted sequencing of 314 nuclear genes encoding respiratory chain complex subunits and proteins implicated in mitochondrial oxidative phosphorylation (OXPHOS). A decrease of 96-bp in the length of the AGK cDNA sequence was detected. Decreases in the oxygen consumption rate (OCR) and the OCR:ECAR (extracellular acidification rate) ratio in the patient’s fibroblasts indicated reduced electron flow through the respiratory chain, and spectrophotometry revealed decreased activity of OXPHOS complexes I and V. We demonstrate a clear defect in mitochondrial function in the patient’s fibroblasts and describe the possible molecular mechanism underlying the pathogenicity of this novel AGK variant. Experimental validation using in vitro analysis allowed an accurate characterization of the disease-causing variant.     

Studies on liver phosphatidyl cholines: II. Effect of sex,age and species differences on phosphatidyl cholines from liver mitochondria and microsomes

Liver mitochondrial and microsomal phosphatidyl cholines differing in the degree of unsaturation of their fatty acids have been separated into four fractions by silver ion silica gel TLC. The levels of the four phosphatidyl choline fractions were determined for male and female rats and mice, fetal and young rabbits, and female hamsters and guinea pigs. The sum of phosphatidyl choline fractions 1, 2, and 3 of mitochondria and microsomes was greater in the female rat than in the male rat with the difference being a reflection of a higher level of fraction 3 which contains arachidonic acid. The female rat has greater concentration of phosphatidyl choline fractions 1 and 3 of mitochondria. Similar results were seen in mouse liver microsomes but not in mitochondria. The levels of the individual four fractions varied from species to species. No change occurred in the levels of the phosphatidyl choline fractions of fetal (−9 and −3 days) rabbits, but an increase was seen in the level of fraction 4 between day 3 and day 35 in both the mitochondria and microsomal fractions of liver. The concentration of mitochondrial and microsomal protein, total phospholipid and total lecithin phosphorus were determined in rat, mouse, hamster and guinea pig. The total phospholipid phosphorus/protein (μg/mg) of microsomes was greater in all species than that observed in mitochondria. Liver microsomes contain 45–50% of total phospholipid phosphorus as lecithin whereas mitochondria contains 32–37%. The fatty acid patterns of mitochondria and microsomal phosphatidyl cholines were determined and the ratio of palmitate to stearate was greater than two for mice and hamsters and approximately 0.5 for rat and guinea pigs.     

Roles of Oxidative Stress, Apoptosis, PGC-1α and Mitochondrial Biogenesis in Cerebral Ischemia

The primary physiological function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Overproduction of reactive oxygen species (ROS) as byproducts generated from mitochondria have been implicated in acute brain injuries such as stroke from cerebral ischemia. It was well-documented that mitochondria-dependent apoptotic pathway involves pro- and anti-apoptotic protein binding, release of cytochrome c, leading ultimately to neuronal death. On the other hand, mitochondria also play a role to counteract the detrimental effects elicited by excessive oxidative stress. Recent studies have revealed that oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves peroxisome proliferative activated receptor-γ (PPARγ) co-activator 1α (PGC1-α). PGC1-α is a master regulator of ROS scavenging enzymes including manganese superoxide dismutase 2 and the uncoupling protein 2, both are mitochondrial proteins, and may contribute to neuronal survival. PGC1-α is also involved in mitochondrial biogenesis that is vital for cell survival. Experimental evidence supports the roles of mitochondrial dysfunction and oxidative stress as determinants of neuronal death as well as endogenous protective mechanisms after stroke. This review aims to summarize the current knowledge focusing on the molecular mechanisms underlying cerebral ischemia involving ROS, mitochondrial dysfunction, apoptosis, mitochondrial proteins capable of ROS scavenging, and mitochondrial biogenesis.     

CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.     

tRNA Modification Enzymes GidA and MnmE: Potential Role in Virulence of Bacterial Pathogens

Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism.     

Identification of the High Molecular Weight Isoform of Phostensin

Phostensin is encoded by KIAA1949. 5′-RACEanalysis has been used to identify the translation start site of phostensin mRNA, indicating that it encodes 165 amino acids with an apparent molecular weight of 26 kDa on SDS-PAGE. This low-molecular-weight phostensin is present in human peripheral blood mononuclear cells and many leukemic cell lines. Phostensin is a protein phosphatase-1(PP1) binding protein. It also contains one actin-binding motif at its C-terminal region and binds to the pointed ends of actin filaments, modulating actin dynamics. In the current study, a high-molecular-weight phostensin is identified by using immunoprecipitationin combination with a proteomic approach. This new species of phostensin is also encoded by KIAA1949 and consists of 613 amino acids with an apparent molecular weight of 110 kDa on SDS-PAGE. The low-molecular-weight and high-molecular-weight phostensins were named as phostensin-α and phostensin-β, respectively. Although phostensin-α is the C-terminal region of phostensin-β, it is not degraded from phostensin-β. Phostensin-β is capable of associating with PP1 and actin filaments, and is present in many cell lines.