Mitochondrial Function In Human Cells Research Articles

Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex.

Despite decades of extensive research, mitochondrial lipid transport is a process far from fully understood. In this issue, Sassano et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206008) identified a new complex, composed of E-Syt1 and PERK, which mediates lipid transport at ER-mitochondria contact sites and regulates mitochondrial functions in human cells.

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.

Depletion of oxaloacetate decarboxylase FAHD1 inhibits mitochondrial electron transport and induces cellular senescence in human endothelial cells

In this study we report the identification of FAH domain containing protein 1 (FAHD1), a recently described member of the fumarylacetoacetate hydrolase (FAH) superfamily of metabolic enzymes, as a novel player in the regulation of cellular senescence. FAHD1 was found in a proteomic screen searching for mitochondrial proteins, which are differentially regulated in mitochondria from young and senescent human endothelial cells, and subsequently identified as oxaloacetate decarboxylase. We report here that depletion of FAHD1 from human endothelial cells inhibited mitochondrial energy metabolism and subsequently induced premature senescence. Whereas senescence induced by FAHD1 depletion was not associated with DNA damage, we noted a reduction of mitochondrial ATP-coupled respiration associated with upregulation of the cdk inhibitor p21. These results indicate that FAHD1 is required for mitochondrial function in human cells and provide additional support to the growing evidence that mitochondrial dysfunction can induce cellular senescence by metabolic alterations independent of the DNA damage response pathway.

Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET

MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane α-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. In the presence of NADH and flavin reductase, 1 molecule of flavin nucleotide is sufficient to reduce about 100 molecules of the mitoNEET [2Fe-2S] clusters in 4min under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that flavin mononucleotide (FMN), but not flavin adenine dinucleotide (FAD), has a specific interaction with mitoNEET. Molecular docking models further reveal that flavin mononucleotide binds mitoNEET at the region between the N-terminal transmembrane α-helix and the [2Fe-2S] cluster binding domain. The closest distance between the [2Fe-2S] cluster and the bound flavin mononucleotide in mitoNEET is about 10Å, which could facilitate rapid electron transfer from the reduced flavin nucleotide to the [2Fe-2S] cluster in mitoNEET. The results suggest that flavin nucleotides may act as electron shuttles to reduce the mitoNEET [2Fe-2S] clusters and regulate mitochondrial functions in human cells.

Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease.

Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-β-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.

Mitochondrial function is impaired in yeast and human cellular models of Shwachman Diamond syndrome

Shwachman Diamond syndrome (SDS) is an inherited bone marrow failure syndrome typically characterized by neutropenia, exocrine pancreas dysfunction, metaphyseal chondrodysplasia, and predisposition to myelodysplastic syndrome and leukemia. SBDS, the gene affected in most cases of SDS, encodes a protein known to influence many cellular processes including ribosome biogenesis, mitotic spindle assembly, chemotaxis, and the regulation of reactive oxygen species production. The best characterized role for the SBDS protein is in the production of functional 60S ribosomal subunits. Given that a reduction in functional 60S subunits could impact on the translational output of cells depleted of SBDS we analyzed protein synthesis in yeast cells lacking SDO1, the ortholog of SBDS. Cells lacking SDO1 selectively increased the synthesis of POR1, the ortholog of mammalian VDAC1 a major anion channel of the mitochondrial outer membrane. Further studies revealed the cells lacking SDO1 were compromised in growth on non-fermentable carbon sources suggesting mitochondrial function was impaired. These observations prompted us to examine mitochondrial function in human cells where SBDS expression was reduced. Our studies indicate that reduced expression of SBDS decreases mitochondrial membrane potential and oxygen consumption and increases the production of reactive oxygen species. These studies indicate that mitochondrial function is also perturbed in cells expressing reduced amounts of SBDS and indicate that disruption of mitochondrial function may also contribute to SDS pathophysiology.

Thyroid hormone effect on human mitochondria measured by flow cytometry

Background: Mitochondrial function may be impaired in a number of diseases including metabolic syndrome, cardiovascular disease and endocrine disorders. Therefore it is important to be able to measure mitochondrial function in human cells. Purpose: The aim of the present study was to evaluate a method to measure mitochondrial function in human derived cells, which also would reflect regulation by thyroid hormones. Methods: The MDA-MB-231 cell line (a human breast cancer cell line) was incubated with bioactive iodothyronines (T4, 3′-3, 5-T3, 3, 5-T2) 50nmol/l for 3 h. Mitochondrial membrane potentials (MMP) were measured by a flow cytometer after staining with Tetramethylrhodamine methyl ester (TMRM). Also, the effect of TRIAC (a stimulator of thyroid hormone nuclear receptors) and L-Carnitine (an inhibitor of thyroid hormone passage into the nucleus) was examined. Findings: It was possible to measure mitochondrial membrane potential (MMP) in human derived cells and to examine thyroid hormone effects using flow cytometry. Bioactive iodothyronines increased mitochondrial membrane potential. TRIAC had no effect and L-Carnitine only inhibited T4 stimulation of membrane potential. Conclusion: Flow cytometry may be a valuable method for examining and testing mitochondrial function in human cells. Our findings demonstrate increase of mitochondrial membrane potential and an extra nuclear short time effect of 3, 5-T2 on mitochondrial activity.