Osteosarcoma is a differentiation-deficient disease, and despite the unique advantages and great potential of differentiation therapy, there are only a few known differentiation inducers, and little research has been done on their targets. Cell differentiation is associated with an increase in mitochondrial content and activity. The metabolism of some tumor cells is characterized by impaired oxidative phosphorylation, as well as up-regulation of aerobic glycolysis and pentose phosphate pathways. Leucine-containing zipper and EF-hand transmembrane protein 1 (LETM1) is involved in the maintenance of mitochondrial morphology and is closely associated with tumorigenesis and progression, as well as cancer cell stemness. We found that MG63 and 143B osteosarcoma cells overexpress LETM1 and exhibit abnormalities in mitochondrial structure and function. Knockdown of LETM1 partially restored the mitochondrial structure and function, inhibited the pentose phosphate pathway, promoted oxidative phosphorylation, and led to osteogenic differentiation. It also inhibited spheroid cell formation, proliferation, migration, and invasion in an in vitro model. When LETM1 was knocked down in vivo, there was reduced tumor formation and lung metastasis. These data suggest that mitochondria are aberrant in LETM1-overexpressing osteosarcoma cells, and knockdown of LETM1 partially restores the mitochondrial structure and function, inhibits the pentose phosphate pathway, promotes oxidative phosphorylation, and increases osteogenic differentiation, thereby reducing malignant biological behavior of the cells.

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.

Previous studies have suggested that circular RNAs (circRNAs) play important regulatory roles in cancer progression. Previous evidence exhibited the aberrant upregulation of circ_0061140 in ovarian cancer. However, the detailed role of circ_0061140 in ovarian cancer progression and its associated mechanism remain largely unknown and need further exploration. The expression of circ_0061140, microRNA-761 (miR-761) and leucine zipper and EF-hand containing transmembrane protein 1 (LETM1) was checked by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or western blot. Cell Counting Kit-8 (CCK8), colony formation, 5-Ethynyl-2\'-deoxyuridine (EdU), flow cytometry, wound healing, transwell, and tube formation assays were conducted to assess cell functions. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the interaction between miR-761 and circ_0061140 or LETM1. Xenograft tumor model was established to analyze the role of circ_0061140 in tumor growth in vivo. Circ_0061140 expression was notably up-regulated in ovarian cancer tissues and cell lines. Circ_0061140 knockdown suppressed the proliferation, migration, invasion, and angiogenesis and triggered the apoptosis of ovarian cancer cells. Circ_0061140 directly interacted with miR-761, and circ_0061140 silencing-mediated anti-tumor effects were partly abolished by miR-761 knockdown in ovarian cancer cells. LETM1 was a direct target of miR-761, and LETM1 overexpression partly counteracted miR-761-induced anti-tumor effects. Circ_0061140 could up-regulate LETM1 expression by sponging miR-761. Circ_0061140 knockdown significantly suppressed xenograft tumor growth in vivo. Circ_0061140 aggravated ovarian cancer progression through miR-761-dependent regulation of LETM1.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Seizures are one of the clinical hallmarks of Wolf-Hirschhorn syndrome (WHS), causing a significant impact on the life quality, still in the first years of life. Even that the knowledge about WHS-related seizure candidate genes has grown, cumulative evidence suggests synergic haploinsufficiency of distinct genes within cellular networks that should be better elucidated. Herein, we evaluated common mechanisms between candidate genes from WHS seizure-susceptibility regions (SSR) and genes globally associated with epilepsy. For this purpose, data from 94 WHS patients delineated by chromosomal microarray analysis were integrated into a tissue-specific gene network with gene expression, drugs, and biological processes. We found functional modules and signaling pathways involving candidate and new genes with potential involvement in the WHS-related seizure phenotype. The proximity among the previous reported haploinsufficient candidate genes (PIGG, CPLX1, CTBP1, LETM1) and disease genes associated with epilepsy suggests not just one, but different impaired mechanisms in cellular networks responsible for the balance of neuronal activity in WHS patients, from which neuron communication is the most impaired in WHS-related seizures. Furthermore, CTBP1 obtained the largest number of drug associations, reinforcing its importance for adaptations of brain circuits and its putative use as a pharmacological target for treating seizures/epilepsy in patients with WHS.

暂无摘要。

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca2+ and K+ homeostasis in mammalian cells. Here, we demonstrate that the Letm1 orthologue of Trypanosoma cruzi, the etiologic agent of Chagas disease, is important for mitochondrial Ca2+ uptake and release. The results show that both mitochondrial Ca2+ influx and efflux are reduced in TcLetm1 knockdown (TcLetm1-KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential. Remarkably, TcLetm1 knockdown or overexpression increases or does not affect mitochondrial Ca2+ levels in epimastigotes, respectively. TcLetm1-KD epimastigotes have reduced growth, and both overexpression and knockdown of TcLetm1 cause a defect in metacyclogenesis. TcLetm1-KD also affected mitochondrial bioenergetics. Invasion of host cells by TcLetm1-KD trypomastigotes and their intracellular replication is greatly impaired. Taken together, our findings indicate that TcLetm1 is important for Ca2+ homeostasis and cell viability in T cruzi.

Mitochondria are dynamic organelles, which serve various purposes, including but not limited to the production of ATP and various metabolites, buffering ions, acting as a signaling hub, etc. In recent years, mitochondria are being seen as the central regulators of cellular growth, development, and death. Since neurons are highly specialized cells with a heavy metabolic demand, it is not surprising that neurons are one of the most mitochondria-rich cells in an animal. At synapses, mitochondrial function and dynamics is tightly regulated by synaptic calcium. Calcium influx during synaptic activity causes increased mitochondrial calcium influx leading to an increased ATP production as well as buffering of synaptic calcium. While increased ATP production is required during synaptic transmission, calcium buffering by mitochondria is crucial to prevent faulty neurotransmission and excitotoxicity. Interestingly, mitochondrial calcium also regulates the mobility of mitochondria within synapses causing mitochondria to halt at the synapse during synaptic transmission. In this review, we summarize the various roles of mitochondrial calcium at the synapse.

[This corrects the article DOI: 10.3389/fphys.2019.00431.].

Leucine Zipper EF-hand containing transmembrane protein-1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca2+ )/proton exchange. The matrix residing carboxyl (C)-terminal domain contains a sequence identifiable EF-hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca2+ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF-hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α-helical secondary structure that is augmented in the presence of Ca2+ . Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca2+ -binding affinity, consistent with the canonical Ca2+ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4-fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF-hand properties, Ca2+ binding increases the exposure of hydrophobic regions, typical of EF-hands; however, this Ca2+ -induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF-hand within LETM1 is unreactive to Ca2+ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca2+ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.