Identification of Mfn2-S249 as a Phosphoregulatory Switch of Mitochondrial Fusion Dynamics

Mitochondrial remodeling is a fundamental process underlying cellular respiration and metabolism. Here we report TAK1 as a direct regulator of mitochondrial fusion. TAK1 is activated by a variety of mitogenic factors, cytokines and environmental stimuli, which we find induces rapid fragmentation through Mfn2 inactivation. TAK1 phosphorylates Mfn2 at Ser249, which inhibits the binding of GTP required for Mfn trans-dimerization and mitochondrial membrane fusion. Accordingly, expression of Mfn2-S249 phosphomimetics (Mfn2-E/D) constitutively promote fission whereas alanine mutant (Mfn2-A) yields hyperfused mitochondria and increased bioenergetics in cells. In mice, Mfn2-E knock-in yields embryonic lethality in homozygotes whereas heterozygotes are viable but exhibit increased visceral fat accumulation despite normal body weight and cognitive/motor functions compared to wildtype and Mfn2-A mice. Mature white adipocytes isolated from mutant mice reveal cell-autonomous TAK1-related effects on mitochondrial remodeling and lipid metabolism. These results identify Mfn2-S249 as a dynamic phosphoregulatory switch of mitochondrial fusion during development and energy homeostasis.

Thymosin beta-4 improves endothelial function and reparative potency of diabetic endothelial cells differentiated from patient induced pluripotent stem cells

Background Prior studies show that signature phenotypes of diabetic human induced pluripotent stem cells derived endothelial cells (dia-hiPSC-ECs) are disrupted glycine homeostasis, increased senescence, impaired mitochondrial function and angiogenic potential as compared with healthy hiPSC-ECs. In the current study, we aimed to assess the role of thymosin β-4 (Tb-4) on endothelial function using dia-hiPSC-ECs as disease model of endothelial dysfunction. Methods and results Using dia-hiPSC-ECs as models of endothelial dysfunction, we determined the effect of Tb-4 on cell proliferation, senescence, cyto-protection, protein expression of intercellular adhesion molecule-1 (ICAM-1), secretion of endothelin-1 and MMP-1, mitochondrial membrane potential, and cyto-protection in vitro and angiogenic potential for treatment of ischemic limb disease in a mouse model of type 2 diabetes mellitus (T2DM) in vivo. We found that 600 ng/mL Tb4 significantly up-regulated AKT activity and Bcl-XL protein expression, enhanced dia-hiPSC-EC viability and proliferation, limited senescence, reduced endothelin-1 and MMP-1 secretion, and improved reparative potency of dia-hiPSC-ECs for treatment of ischemic limb disease in mice with T2DM. However, Tb4 had no effect on improving mitochondrial membrane potential and glycine homeostasis and reducing intercellular adhesion molecule-1 protein expression in dia-hiPSC-ECs. Conclusions Tb-4 improves endothelial dysfunction through enhancing hiPSC-EC viability, reducing senescence and endothelin-1 production, and improves angiogenic potency in diabetes.

The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H+ transporter activated by NH3 and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H+ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11’s function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some “glutamine-addicted” cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.

SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment

Mitochondria, which is essential for adequate innate immune response, energy metabolism and mitochondria reactive oxygen species (ROS) production, might be in the cross fire of Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host cell defense. However, little is known about interactions between mitochondria and SARS-CoV-2. We performed fluorescent microscopy and found an enrichment of SARS-CoV-2 replication products double stranded RNA (dsRNA) within mitochondria. The entry process of dsRNA might be mediated by Tom20 as observed by reduced mitochondrial localization of SARS-CoV-2 dsRNA in Tom20 knockdown cells. Importantly, decreased mitochondrial localization of dsRNA, as well as mitochondrial membrane stabilizers mdivi-1 and cyclosporin A, inhibited viral load in cells. Next, we detected mitochondrial dysfunction caused by SARS-CoV-2 infection, including mitochondrial membrane depolarization, mitochondrial permeability transition pore opening and increased ROS release. In response to mitochondrial damage, we observed an increase in expression and mitochondrial accumulation of Pink1 and Parkin proteins, as well as Pink-1-mediated recruitment of P62 to mitochondria, suggesting initiated mitophagy for mitochondrial quality control and virus clearance. Nevertheless, we observed that mitophagy was inhibited and stayed in early stage with an unchanged Hsp60 expression post SARS-CoV-2 infection. This might be one of the anti-autophagy strategies of SARS-CoV-2 and we used co-immunoprecipitation to found that SARS-CoV-2 infection inhibited P62 and LC3 binding which plays a critical role in selective envelopment of substrates into autophagosomes. Our results suggest that mitochondria are closely involved in SARS-CoV-2 replication and mitochondrial homeostasis is disrupted by SARS-CoV-2 in the virus-cell confrontation.

Nisin induces apoptosis in cervical cancer cells via reactive oxygen species generation and mitochondrial membrane potential changes

Nisin, an antimicrobial peptide produced by Lactococcus lactis, is widely used as a safe food preservative and has been recently attracting the attention of many researchers as a potential anticancer agent. The cytotoxicity of nisin against HeLa, OVCAR-3, SK-OV-3, and HUVEC cells was evaluated using MTT assay. The apoptotic effect of nisin was identified by Annexin-V/propidium iodide assay, and then it was further confirmed by western blotting analysis, mitochondrial membrane potential (ΔΨm) analysis, and reactive oxygen species (ROS) assay. The MTT assay showed concentration-dependent cytotoxicity of nisin towards cancer cell lines, with the IC50 values of 11.5-23 µM, but less toxicity against normal endothelial cells. Furthermore, treatment of cervical cancer cells with 12 µM nisin significantly (P<0.05) increased the Bax/Bcl-2 ratio (4.9-fold), reduced ΔΨm (70%), and elevated ROS levels (1.7-fold). These findings indicated that nisin might have anticancer and apoptogenic activities through mitochondrial dysfunction and oxidative stress damage in cervical cancer cells.

The Metaxin Mitochondrial Import Proteins: Multiple Metaxin-like Proteins in Fungi

Multiple metaxin-like proteins are shown to exist in fungi, as also found for the metaxin proteins of vertebrates and invertebrates. In vertebrates, metaxins 1 and 2 are mitochondrial membrane proteins that function in the import of proteins into mitochondria. Fungal metaxin-like proteins were identified by criteria including their homology with human metaxins and the presence of characteristic GST_N_Metaxin, GST_C_Metaxin, and Tom37 protein domains. Fungi in different taxonomic divisions (phyla) were found to possess multiple metaxin-like proteins. These include the Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Mucoromycota, Neocallimastigomycota, and Zoopagomycota divisions. Most fungi with multiple metaxin-like proteins contain two proteins, designated MTXa and MTXb. Amino acid sequence alignments show a high degree of homology among MTXa proteins, with over 60% amino acid identities, and also among MTXb proteins of fungi in the same division. But very little homology is observed in aligning MTXa with MTXb proteins of the same or different fungi. Both the MTXa proteins and MTXb proteins have the protein domains that characterize the metaxins and metaxin-like proteins: GST_N_Metaxin, GST_C_Metaxin, and Tom37. The metaxins and metaxin-like proteins of vertebrates, invertebrates, plants, protists, and bacteria all possess these domains. The secondary structures of MTXa and MTXb proteins are both dominated by similar patterns of α-helical segments, but extensive β-strand segments are absent. Nine highly conserved α-helical segments are present, the same as other metaxins and metaxin-like proteins. Phylogenetic analysis reveals that MTXa and MTXb proteins of fungi form two separate and distinct groups. These groups are also separate from the groups of vertebrate metaxins, metaxin-related Sam37 proteins of yeasts, and metaxin-like FAXC proteins.

Stepwise Cerebral Ischemia Causes Disturbances in Mitochondrial Respiration of Neurons in the Cerebral Cortex of Rats

Objectives: To conduct a comparative analysis of respiration of mitochondria of brain homogenates of rats with stepwise subtotal cerebral ischemia with different duration between ligations of both common carotid arteries. Methods: The experiments were performed on 24 male mongrel white rats weighing 260 ±20 g. Cerebral ischemia (CI) was simulated under intravenous thiopental anesthesia (40-50 mg/kg). The control group consisted of falsely operated rats of similar sex and weight. To study mitochondrial respiration, the brain was extracted in the cold (0-4°C), dried with filter paper, weighed and homogenized in an isolation medium containing 0.32 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 (in a ratio of 1:10), using Potter-Evelheim homogenizer with Teflon pestle according to the modified method. To prevent systematic measurement errors, brain samples from the compared control and experimental groups of animals were studied under the same conditions. Results: Stepwise SCI with an interval of 1 and 3 days between bandages of both OCA leads to damage to the neurons of the parietal cortex and hippocampus of rats, which manifests itself in a decrease in their size, deformation of the pericaryons, an increase in the number of shrunken neurons and shadow cells. The most pronounced changes were observed in the subgroup with an interval between dressings of 1 day. These changes were similar to the changes in SCI (p>0.05), except for the absence of cells with pericellular edema in the hippocampus and a smaller number of them in the parietal cortex. SCI with an interval between WASP dressings of 7 days, on the contrary, it is manifested by less pronounced histological changes, especially in the hippocampus. Conclusion: In cerebral ischemia, damage to the inner mitochondrial membrane occurs due to activation of free radical oxidation processes. Damage to the inner mitochondrial membrane, in turn, leads to an increase in its permeability and a decrease in the level of the proton gradient due to the transition of protons along the concentration gradient through the resulting nonspecific pores into the mitochondrial matrix. As a result, the efficiency of ATP synthesis decreases, and more substrates and oxygen are required to maintain the intermembrane potential under these conditions.

Mitochondrial Membrane Remodeling

Mitochondria are key regulators of many important cellular processes and their dysfunction has been implicated in a large number of human disorders. Importantly, mitochondrial function is tightly linked to their ultrastructure, which possesses an intricate membrane architecture defining specific submitochondrial compartments. In particular, the mitochondrial inner membrane is highly folded into membrane invaginations that are essential for oxidative phosphorylation. Furthermore, mitochondrial membranes are highly dynamic and undergo constant membrane remodeling during mitochondrial fusion and fission. It has remained enigmatic how these membrane curvatures are generated and maintained, and specific factors involved in these processes are largely unknown. This review focuses on the current understanding of the molecular mechanism of mitochondrial membrane architectural organization and factors critical for mitochondrial morphogenesis, as well as their functional link to human diseases.

Lipopolysaccharide Enhances Genotoxicity by Activating GADD45G and NF-κB in Human Corneal Epithelial Cells

As the prevalence of microbial keratitis increases, it creates an environment conducive to genotoxicity response. A potential connection between growth arrest and DNA-damage-inducible 45 gamma (GADD45G) gene expression has not been proven in the corneal epithelial cells. The aim of this study was to determine whether lipopolysaccharide (LPS) enhances genotoxicity, DNA damage, and inflammatory responses in human corneal epithelial cells (HCECs) in vitro. In a set of parameters, cytotoxicity, reactive oxygen species, mitochondrial membrane potential, DNA damage, inflammatory response, and apoptosis were assessed. LPS (1, 5, and 10 μg/mL) treated HCECs were increased reactive oxygen species formation, mitochondrial membrane depolarization, and genotoxicity in a concentration-dependent manner. Similarly, NF-κB, PARP1, and TP53 were also overexpressed in the LPS treated HCECs. 24 hours after LPS induction, micronucleus scoring, and proapoptotic factors were also increased. Among them, the GADD45G, NF-κB, and γH2AX were overexpressed both on the mRNA and protein levels in LPS (10 μg/mL) treated HCECs. In our study, we show that the GADD45G signaling can trigger genotoxic instability in HCECs exposed to LPS. Therefore, understanding the factors contributing to infectious keratitis, such as GADD45G, NF-κB, and γH2AX signaling, may help to develop antigenotoxic and anti-inflammatory therapies for corneal dystrophy and epithelial cell remodeling.