LETM1: A Single Entity With Diverse Impact on Mitochondrial Metabolism and Cellular Signaling.
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ABSTRACT:
SUBMITTER: Natarajan GK
PROVIDER: S-EPMC8012663 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dysregulation of mitochondrial Ca(2+)-dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca(2+) transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca(2+) transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca(2+) transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca(2+) influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ΔEF hand, (D676A D688K)LETM1 mutant-overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS-derived fibroblasts, the mitochondrial Ca(2+) uniporter components MCU, MCUR1, and MICU1 remain unaltered. In addition, the MCU mitoplast patch-clamp current (IMCU) was largely unaffected in LETM1-knockdown cells. Silencing of LETM1 also impaired basal mitochondrial oxygen consumption, possibly via complex IV inactivation and ATP production. Remarkably, LETM1 knockdown also resulted in increased reactive oxygen species production. Further, LETM1 silencing promoted AMPK activation, autophagy, and cell cycle arrest. Reconstitution of LETM1 or antioxidant overexpression rescued mitochondrial Ca(2+) transport and bioenergetics. These findings reveal the role of LETM1-dependent mitochondrial Ca(2+) flux in shaping cellular bioenergetics.
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Project description:The diverse clinical phenotypes of Wolf-Hirschhorn syndrome (WHS) are the result of haploinsufficiency of several genes, one of which, LETM1, encodes a protein of the mitochondrial inner membrane of uncertain function. Here, we show that LETM1 is associated with mitochondrial ribosomes, is required for mitochondrial DNA distribution and expression, and regulates the activity of an ancillary metabolic enzyme, pyruvate dehydrogenase. LETM1 deficiency in WHS alters mitochondrial morphology and DNA organization, as does substituting ketone bodies for glucose in control cells. While this change in nutrient availability leads to the death of fibroblasts with normal amounts of LETM1, WHS-derived fibroblasts survive on ketone bodies, which can be attributed to their reduced dependence on glucose oxidation. Thus, remodeling of mitochondrial nucleoprotein complexes results from the inability of mitochondria to use specific substrates for energy production and is indicative of mitochondrial dysfunction. However, the dysfunction could be mitigated by a modified diet-for WHS, one high in lipids and low in carbohydrates.
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Project description:The diverse clinical phenotypes of Wolf-Hirschhorn syndrome (WHS) are the result of haploinsufficiency of several genes, one of which, LETM1, encodes a protein of the mitochondrial inner membrane of uncertain function. Here we show that LETM1 is associated with mitochondrial ribosomes, is required for mitochondrial DNA distribution and expression, and regulates the activity of an ancillary metabolic enzyme, pyruvate dehydrogenase. LETM1 deficiency in WHS alters mitochondrial morphology and DNA organization, as does substituting ketone bodies for glucose in control cells. While this change in nutrient availability leads to the death of fibroblasts with normal amounts of LETM1, WHS-derived fibroblasts survive on ketone bodies, which can be attributed to their reduced dependence on glucose oxidation. Thus, remodeling of mitochondrial nucleoprotein complexes results from the inability of mitochondria to use specific substrates for energy production and is indicative of mitochondrial dysfunction. However, the dysfunction could be mitigated by a modified diet - for WHS one high in lipids and low in carbohydrates.
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Project description:Cilia are organelles specialized in movement and signal transduction. The ciliary transient receptor potential ion channel polycystin-2 (TRPP2) controls elementary cilia-mediated physiological functions ranging from male fertility and kidney development to left-right patterning. However, the molecular components translating TRPP2 channel-mediated Ca2+ signals into respective physiological functions are unknown. Here, we show that the Ca2+-regulated mitochondrial ATP-Mg/Pi solute carrier 25 A 25 (SLC25A25) acts downstream of TRPP2 in an evolutionarily conserved metabolic signaling pathway. We identify SLC25A25 as an essential component in this cilia-dependent pathway using a genome-wide forward genetic screen in Drosophila melanogaster, followed by a targeted analysis of SLC25A25 function in zebrafish left-right patterning. Our data suggest that TRPP2 ion channels regulate mitochondrial SLC25A25 transporters via Ca2+ establishing an evolutionarily conserved molecular link between ciliary signaling and mitochondrial metabolism.
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Project description:The leucine zipper, EF hand-containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca(2+) and K(+) homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca(2+) fluorophore and (45)Ca(2+)-based assays, we demonstrate directly that Letm1 is a Ca(2+) transporter, with apparent affinities of cations in the sequence of Ca(2+) ? Mn(2+) > Gd(3+) ? La(3+) > Sr(2+) >> Ba(2+), Mg(2+), K(+), Na(+). Kinetic analysis yields a Letm1 turnover rate of 2 Ca(2+)/s and a Km of ?25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca(2+)/2 H(+) antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H(+)-dependent Ca(2+) transport mechanism identified in intact mitochondria.
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Project description:Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. 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.
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Project description:Mitochondrial metabolism, respiration, and ATP production necessitate ion transport across the inner mitochondrial membrane. Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1), one of the genes deleted in Wolf-Hirschhorn syndrome, encodes a putative mitochondrial Ca(2+)/H(+) antiporter. Cellular Letm1 knockdown reduced Ca(2+)mito uptake, H(+)mito extrusion and impaired mitochondrial ATP generation capacity. Homozygous deletion of Letm1 in mice resulted in embryonic lethality before day 6.5 of embryogenesis and ~50% of the heterozygotes died before day 13.5 of embryogenesis. The surviving heterozygous mice exhibited altered glucose metabolism, impaired control of brain ATP levels, and increased seizure activity. We conclude that loss of Letm1 contributes to the pathology of Wolf-Hirschhorn syndrome in humans and may contribute to seizure phenotypes by reducing glucose oxidation and other specific metabolic alterations.
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Project description:Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson's disease (PD). PINK1 loss is associated with mitochondrial Ca2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP+-MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca2+ transport..
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