The essential role of adenine nucleotide translocase 4 on male reproductive function in mice.

Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.

The essential role of adenine nucleotide translocase 4 on male reproductive function in mice

Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.

GRP75 Regulates Mitochondrial-Supercomplex Turnover to Modulate Insulin Sensitivity.

GRP75 (75-kDA glucose-regulated protein), defined as a major component of both the mitochondrial quality control system and mitochondria-associated membrane, plays a key role in mitochondrial homeostasis. In this study, we assessed the roles of GRP75, other than as a component, in insulin action in both in vitro and in vivo models with insulin resistance. We found that GRP75 was downregulated in mice fed a high-fat diet (HFD) and that induction of Grp75 in mice could prevent HFD-induced obesity and insulin resistance. Mechanistically, GRP75 influenced insulin sensitivity by regulating mitochondrial function through its modulation of mitochondrial-supercomplex turnover rather than mitochondria-associated membrane communication: GRP75 was negatively associated with respiratory chain complex activity and was essential for mitochondrial-supercomplex assembly and stabilization. Moreover, mitochondrial dysfunction in Grp75-knockdown cells might further increase mitochondrial fragmentation, thus triggering cytosolic mtDNA release and activating the cGAS/STING-dependent proinflammatory response. Therefore, GRP75 can serve as a potential therapeutic target of insulin resistant-related diabetes or other metabolic diseases.

Iron-sulphur cluster biogenesis factor LYRM4 is a novel prognostic biomarker associated with immune infiltrates in hepatocellular carcinoma.

BACKGROUND: LYRM4 is necessary to maintain the stability and activity of the human cysteine desulfurase complex NFS1-LYRM4-ACP. The existing experimental results indicate that cancer cells rely on the high expression of NFS1. However, the role of LYRM4 in liver hepatocellular carcinoma (LIHC) remains unclear. METHODS: In this study, we combined bioinformatics analysis and clinical specimens to evaluate the mRNA, protein expression, and gene regulatory network of LYRM4 in LIHC. Furthermore, we detected the activity of several classical iron-sulphur proteins in LIHC cell lines through UV-vis spectrophotometry. RESULTS: The mRNA and protein levels of LYRM4 were upregulated in LIHC. Subsequent analysis revealed that the LYRM4 mRNA expression was related to various clinical stratifications, prognosis, and survival of LIHC patients. In addition, the mRNA expression of LYRM4 was significantly associated with ALT, tumour thrombus, and encapsulation of HBV-related LIHC patients. IHC results confirmed that LYRM4 was highly expressed in LIHC tissues and showed that the expression of LYRM4 protein in LIHC was significantly correlated with age and serum low-density lipoprotein (LDL) and triglyceride (TG) content. In particular, the mRNA expression of key iron- sulphur proteins POLD1 and PRIM2 was significantly overexpressed and correlated with poor prognosis in LIHC patients. Compared with hepatocytes, the activities of mitochondrial complex I and aconitate hydratase (ACO2) in LIHC cell lines were significantly increased. These results indicated that the iron-sulphur cluster (ISC) biosynthesis was significantly elevated in LIHC, leading to ISC-dependent metabolic reprogramming. Changes in the activity of ISC-dependent proteins may also occur in paracancerous tissues. Further analysis of the biological interaction and gene regulation networks of LYRM4 suggested that these genes were mainly involved in the citric acid cycle and oxidative phosphorylation. Finally, LYRM4 expression in LIHC was significantly positively correlated with the infiltrating levels of six immune cell types, and both factors were strongly associated with prognosis. CONCLUSION: LYRM4 could be a novel prognostic biomarker and molecular target for LIHC therapy. In particular, the potential regulatory networks of LYRM4 overexpression in LIHC provide a scientific basis for future research on the role of the ISC assembly mechanism and LYRM4-mediated sulphur transfer routes in carcinogenesis.

Assessment of mitochondrial function in metabolic dysfunction-associated fatty liver disease using obese mouse models.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is characterized by deregulated hepatic lipid metabolism; however, the association between MAFLD development and mitochondrial dysfunction has yet to be confirmed. Herein, we employed high-resolution respirometry, blue native polyacrylamide gel electrophoresis-based in-gel activity measurement and immunoblot analysis to assess mitochondrial function in obesity-induced mouse models with varying degrees of MAFLD. Results showed a slight but significant decrease in hepatic mitochondrial respiration in some MAFLD mice compared to mice fed a standard diet. However, the activities and levels of mitochondrial oxidative phosphorylation complexes remained unchanged during obesity-induced MAFLD progression. These results suggest that mitochondrial function, particularly oxidative phosphorylation, was mildly affected during obesity-induced MAFLD development. Moreover, transcriptome profiling of mouse and human liver tissues with varying degrees of MAFLD revealed that the decreased activation of mitochondria-related pathways was only associated with MAFLD of a high histological grade, whereas the major regulators of mitochondrial biogenesis were not altered in mice or humans during MAFLD development. Collectively, our results suggest that impaired hepatic mitochondrial function is not closely associated with obesity-induced MAFLD. Therefore, therapeutic strategies targeting mitochondria for the treatment of MAFLD should be reconsidered.

A simple and rapid protein purification method based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease.

The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.

Removal of Chromium (VI) by Escherichia coli Cells Expressing Cytoplasmic or Surface-Displayed ChrB: a Comparative Study.

Various genetically engineered microorganisms have been developed for the removal of heavy metal contaminants. Metal biosorption by whole-cell biosorbents can be enhanced by overproduction of metal-binding proteins/peptides in the cytoplasm or on the cell surface. However, few studies have compared the biosorption capacity of whole cells expressing intracellular or surface-displayed metal-adsorbing proteins. In this study, several constructs were prepared for expressing intracellular and surface-displayed Ochrobactrum tritici 5bvl1 ChrB in Escherichia coli BL21(DE3) cells. E. coli cells expressing surface-displayed ChrB removed more Cr(VI) from aqueous solutions than cells with cytoplasmic ChrB under the same conditions. However, intracellular ChrB was less susceptible to variation in extracellular conditions (pH and ionic strength), and more effectively removed Cr(VI) from industrial wastewater than the surface-displayed ChrB at low pH (<3). An adsorptiondesorption experiment demonstrated that compared with intracellular accumulation, cell-surface adsorption is reversible, which allows easy desorption of the adsorbed metal ions and regeneration of the bioadsorbent. In addition, an intrinsic ChrB protein fluorescence assay suggested that pH and salinity may influence the Cr(VI) adsorption capacity of ChrB-expressing E. coli cells by modulating the ChrB protein conformation. Although the characteristics of ChrB may not be universal for all metal-binding proteins, our study provides new insights into different engineering strategies for whole-cell biosorbents for removing heavy metals from industrial effluents.

Systematic analysis of a mitochondrial disease-causing ND6 mutation in mitochondrial deficiency.

BACKGROUND: The m.14487T>C mutation is recognized as a diagnostic mutation of mitochondrial disease during the past 16 years, emerging evidence suggests that mutant loads of m.14487T>C and disease phenotype are not closely correlated. METHODS: Immortalized lymphocytes were generated by coculturing the Epstein-Barr virus and lymphocytes from m.14487T>C carrier Chinese patient with Leigh syndrome. Fifteen cytoplasmic hybrid (cybrid) cell lines were generated by fusing mtDNA lacking 143B cells with platelets donated by patients. Mitochondrial function was systematically analyzed at transcriptomic, metabolomic, and biochemical levels. RESULTS: Unlike previous reports, we found that the assembly of mitochondrial respiratory chain complexes, mitochondrial respiration, and mitochondrial OXPHOS function was barely affected in cybrid cells carrying homoplastic m.14487T>C mutation. Mitochondrial dysfunction associated transcriptomic and metabolomic reprogramming were not detected in cybrid carrying homoplastic m.14487T>C. However, we found that mitochondrial function was impaired in patient-derived immortalized lymphocytes. CONCLUSION: Our data revealed that m.14487T>C mutation is insufficient to cause mitochondrial deficiency; additional modifier genes may be involved in m.14487T>C-associated mitochondrial disease. Our results further demonstrated that a caution should be taken by solely use of m.14487T>C mutation for molecular diagnosis of mitochondrial disease.

Redox regulation by SOD2 modulates colorectal cancer tumorigenesis through AMPK-mediated energy metabolism.

Colorectal cancer (CRC) is a common malignancy. Many reports have implicated aberrant mitochondrial activity in the progression of CRC, with particular emphasis on the dysregulation of redox signaling and oxidative stress. In this study, we focused on manganese superoxide dismutase (MnSOD/SOD2), a key antioxidant enzyme, which maintains intracellular redox homeostasis. Current literature presents conflicting mechanisms for how SOD2 influences tumorigenesis and tumor progression. Here, we explored the role of SOD2 in CRC specifically. We found high levels of SOD2 expression in CRC tissues. We carried out a series of experiments to determine whether knockdown of SOD2 expression in CRC cell lines would reverse features of tumorigenesis. We found that reduced SOD2 expression decreased cell proliferation, migration, and invasion activity in CRC cells. Results from an additional series of experiments on mitochondrial function implicated a dual role for SOD2 in promoting CRC progression. First, proper level of SOD2 helped CRC cells maintain mitochondrial function by disposal of superoxide (O2.- ). Second, over-expression of SOD2 induced H2 O2 -mediated tumorigenesis by upregulating AMPK and glycolysis. Our results indicate that SOD2 may promote the occurrence and development of CRC by regulating the energy metabolism mediated by AMPK signaling pathways.

Mutations in FASTKD2 are associated with mitochondrial disease with multi-OXPHOS deficiency.

Mutations in FASTKD2, a mitochondrial RNA binding protein, have been associated with mitochondrial encephalomyopathy with isolated complex IV deficiency. However, deficiencies related to other oxidative phosphorylation system (OXPHOS) complexes have not been reported. Here, we identified three novel FASTKD2 mutations, c.808_809insTTTCAGTTTTG, homoplasmic mutation c.868C>T, and heteroplasmic mutation c.1859delT/c.868C>T, in patients with mitochondrial encephalomyopathy. Cell-based complementation assay revealed that these three FASTKD2 mutations were pathogenic. Mitochondrial functional analysis revealed that mutations in FASTKD2 impaired the mitochondrial function in patient-derived lymphocytes due to the deficiency in multi-OXPHOS complexes, whereas mitochondrial complex II remained unaffected. Consistent results were also found in human primary muscle cell and zebrafish with knockdown of FASTKD2. Furthermore, we discovered that FASTKD2 mutation is not inherently associated with epileptic seizures, optic atrophy, and loss of visual function. Alternatively, a patient with FASTKD2 mutation can show sinus tachycardia and hypertrophic cardiomyopathy, which was partially confirmed in zebrafish with knockdown of FASTKD2. In conclusion, both in vivo and in vitro studies suggest that loss of function mutation in FASTKD2 is responsible for multi-OXPHOS complexes deficiency, and FASTKD2-associated mitochondrial disease has a high degree of clinical heterogenicity.

Mutations in TOMM70 lead to multi-OXPHOS deficiencies and cause severe anemia, lactic acidosis, and developmental delay.

TOM70 is a member of the TOM complex that transports cytosolic proteins into mitochondria. Here, we identified two compound heterozygous variants in TOMM70 [c.794C>T (p.T265M) and c.1745C>T (p.A582V)] from a patient with severe anemia, lactic acidosis, and developmental delay. Patient-derived immortalized lymphocytes showed decreased TOM70 expression, oligomerized TOM70 complex, and TOM 20/22/40 complex compared with expression in control lymphocytes. Functional analysis revealed that patient-derived cells exhibited multi-oxidative phosphorylation system (OXPHOS) complex defects, with complex IV being primarily affected. As a result, patient-derived cells grew slower in galactose medium and generated less ATP and more extracellular lactic acid than did control cells. In vitro cell model compensatory experiments confirmed the pathogenicity of TOMM70 variants since only wild-type TOM70, but not mutant TOM70, could restore the complex IV defect and TOM70 expression in TOM70 knockdown U2OS cells. Altogether, we report the first case of mitochondrial disease-causing mutations in TOMM70 and demonstrate that TOM70 is essential for multi-OXPHOS assembly. Mutational screening of TOMM70 should be employed to identify mitochondrial disease-causing gene mutations in the future.

Upregulation of MicroRNA-21 promotes tumorigenesis of prostate cancer cells by targeting KLF5.

Prostate cancer (PCa) is the second frequently newly diagnosed cancer in men. Androgen deprivation therapy has been widely used to inhibit PCa growth but eventually fails in many patients. Androgen receptor and its downstream molecules like microRNAs could be promising therapeutic targets. We aimed to investigate the involvement of miR-21 in PCa tumorigenesis. We found that miR-21 was an unfavorable factor and correlated positively with tumor grade in PCa patients from TCGA database. MiR-21 was more highly expressed in androgen-independent PCa cells than in androgen-dependent PCa cells. Overexpression of miR-21 promoted androgen-dependent and -independent PCa cell proliferation, migration, invasion, and resistance to apoptosis. Furthermore, increased miR-21 expression promoted mouse xenograft growth. We identified nine genes differentially expressed in PCa tumors and normal tissue which could be potential targets of miR-21 by bioinformatic analyses. We demonstrate that miR-21 directly targeted KLF5 and inhibited KLF5 mRNA and protein levels in PCa. STRING and functional enrichment analysis results suggest that GSK3B might be regulated by KLF5. Our findings demonstrate that miR-21 promotes the tumorigenesis of PCa cells by directly targeting KLF5. These biological effects are mediated through upregulation of GSK3B and activation of the AKT signaling pathway.

Zinc Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli.

While zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases in Escherichia coli Here, we report that the intracellular zinc overload in E. coli cells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene cluster iscSUA-hscBA-fdx-iscX in E. coli cells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of the E. coli mutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin in E. coli cells.IMPORTANCE Zinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload in Escherichia coli cells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

Oncocytic tumors are marked by enhanced mitochondrial content and mtDNA mutations of complex I in Chinese patients.

Oncocytic tumors are composed of oncocytes characterized by acidophilic granular and reticular cytoplasm. Such features have been attributed to the distinctive aggregation of abnormal mitochondria. Sporadic mitochondrial DNA (mtDNA) mutations, particularly those in complex I subunit genes, have been identified as one of the most noticeable alterations. We reviewed 11,051 cases of patients with thyroid tumors who visited the First Affiliated Hospital of Wenzhou Medical University from January 2011 to August 2017, and we were able to identify 123 cases as oncocytic tumors. We found that older people are at higher risk (P < 0.001) for oncocytic tumors. We confirmed an increased mitochondrial mass in representative samples. Furthermore, a comprehensive analysis of the mitochondrial genomes in patients with oncocytomas revealed 1) haplogroups D5 and A exhibit increased risk of oncocytomas; 2) 60% of mtDNA mutations are in genes encoding respiratory complex subunits while 8% occur in rRNA and 4% in tRNA regions; 3) among mutations in coding regions, 50% are in Complex I genes, including most of the disruptive mutations; 4) 64% of mtDNA mutations are heteroplasmic. Our studies imply a tumorigenesis mechanism for oncocytomas involving mitochondrial alterations mediated by genome instability and modified by mitochondrial haplogroups.

Development of a Sensitive Escherichia coli Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments.

The whole-cell bioreporters based on the cop-operon sensing elements have been proven specifically useful in the assessment of bioavailable copper ions in water environments. In this study, a series of experiments was conducted to further improve the sensitivity and robustness of bioreporters. First, an Escherichia coli △copA△cueO△cusA mutant with three copper transport genes knocked out was constructed. Then, the copAp::gfpmut2 sensing element was inserted into the chromosome of E. coli △copA△cueO△cusA by gene knock-in method to obtain the bioreporter strain E. coli WMC-007. In optimized assay conditions, the linear detection range of Cu2+ was 0.025-5 mg/L (0.39-78.68 µM) after incubating E. coli WMC-007 in Luria-Bertani medium for 5 h. The limit of detection of Cu2+ was 0.0157 mg/L (0.25 µM). Moreover, fluorescence spectrometry and flow cytometry experiments showed more environmental robustness and lower background fluorescence signal than those of the sensor element based on plasmids. In addition, we found that the expression of GFPmut2 in E. coli WMC-007 was induced by free copper ions, rather than complex-bound copper, in a dose-dependent manner. Particularly, the addition of 40 mM 3-(N-Morpholino)propanesulfonic acid buffer to E. coli WMC-007 culture enabled accurate quantification of bioavailable copper content in aqueous solution samples within a pH range from 0.87 to 12.84. The copper recovery rate was about 95.88-113.40%. These results demonstrate potential applications of E. coli WMC-007 as a bioreporter to monitor copper contamination in acidic mine drainage, industrial wastewater, and drinking water. Since whole-cell bioreporters are relatively inexpensive and easy to operate, the combination of this method with other physicochemical techniques will in turn provide more specific information on the degree of toxicity in water environments.

Hepatitis B virus infection is not associated with fatty liver disease: Evidence from a cohort study and functional analysis.

Chronic hepatitis B virus (HBV) infection has been reported to be associated with the prevalence of non­alcoholic fatty liver disease (NAFLD). However, the present study demonstrated that the incidence of fatty liver disease in HBV­infected subjects (16/152, 10.5%) was not significantly different from in non­HBV­infected subjects (292/1,714, 17%), following adjustment for age (odds ratio=0.656; 95% confidence interval=0.379­1.134; P=0.131). Hepatitis B protein X (HBx) is considered a key regulator in HBV infection and several studies have confirmed that HBx serves a pivotal role in the process of fatty liver disease. In the present study, it was demonstrated that HBx­expressing cells exhibited increased mitochondrial membrane potential, ATP generation, and endogenous mitochondrial respiration. In addition, higher levels of mitochondrial reactive oxygen species (ROS) were detected in HBx­expressing cells compared with in control cells. Increased ROS production may contribute to increased lipid droplet formation in HBx­expressing cells, whereas the removal of ROS with N­acetylcysteine may decrease the accumulation of lipid droplets in a time­dependent manner. In conclusion, the present findings indicated that HBV, and perhaps more specifically HBx, was not a protective factor against NAFLD. HBx may function as a risk factor for fatty liver disease, based on the findings of the present functional study; however, further studies are required to clarify the effects of HBx on hepatic steatosis.

Enhanced ROS production leads to excessive fat accumulation through DAF-16 in Caenorhabditis elegans.

Growing evidence shows that enhanced reactive oxygen species (ROS) production is an important contributor to obesity and its co-morbidities, but the functional link between ROS and obesity remains elusive. In this study we used the model animal Caenorhabditis elegans to explore the role of ROS in obesity. Initially, when ROS production was enhanced by treatment with low concentration of paraquat or juglone, both abnormal high fat accumulation and fatty acid composition were observed in wild type worms. We found that the abnormal fat accumulation was associated with increased expression of fat-5, which encodes an isoform of stearoyl-CoA synthetase, and which is regulated by daf-16 encoding the forkhead transcription factor and being activated by downregulation daf-2. When mutant daf-16 worms were used, the abnormal fat accumulation induced by ROS was suppressed. Collectively, we demonstrate that enhanced ROS production can lead to excessive fat accumulation and the change of fatty acid composition. This abnormal phenomenon at least in part depends on the daf-16 pathway by which fat-5 was regulated. The results point towards a role of ROS in obesity in the context of important conserved signaling pathway, thereby guide further studies and future therapeutic interventions.

Dataset of mitochondrial genome variants in oncocytic tumors.

This dataset presents the mitochondrial genome variants associated with oncocytic tumors. These data were obtained by Sanger sequencing of the whole mitochondrial genomes of oncocytic tumors and the adjacent normal tissues from 32 patients. The mtDNA variants are identified after compared with the revised Cambridge sequence, excluding those defining haplogroups of our patients. The pathogenic prediction for the novel missense variants found in this study was performed with the Mitimpact 2 program.

Mitoferrin-1 is Involved in the Progression of Alzheimer's Disease Through Targeting Mitochondrial Iron Metabolism in a Caenorhabditis elegans Model of Alzheimer's Disease.

In mammals, mitoferrin-1 and mitoferrin-2, two homologous proteins of the mitochondrial solute carrier family are required for iron delivery into mitochondria. However, there is only one kind, called W02B12 (mitoferrin-1 or mfn-1), in Caenorhabditis elegans and its regulatory mechanism is unknown. In this study, we used C. elegans strains CL2006 and GMC101 as models to investigate what role mitoferrin-1 played in Alzheimer's disease (AD). We found that knockdown of mitoferrin-1 by feeding-RNAi treatment extended lifespans of both strains of C. elegans. In addition, it reduced the paralysis rate in the GMC101 strain. These results suggest that mitoferrin-1 may be involved in the progression of Alzheimer's disease. Knockdown of mitoferrin-1 was seen to disturb mitochondrial morphology in the CB5600 strain. We tested whether knockdown of mitoferrin-1 could influence mitochondrial metabolism. Analysis of mitochondrial iron metabolism and mitochondrial ROS showed that knockdown of mitoferrin-1 could reduce mitochondrial iron content and reduce the level of mitochondrial ROS in the CL2006 and GMC101 strains. These results confirm that knockdown of mitoferrin-1 can slow the progress of disease in Alzheimer model of C. elegans and suggest that mitoferrin-1 plays a major role in mediating mitochondrial iron metabolism in this process.

mtDNA Haplogroup N9a Increases the Risk of Type 2 Diabetes by Altering Mitochondrial Function and Intracellular Mitochondrial Signals.

Mitochondrial DNA (mtDNA) haplogroups have been associated with the incidence of type 2 diabetes (T2D); however, their underlying role in T2D remains poorly elucidated. Here, we report that mtDNA haplogroup N9a was associated with an increased risk of T2D occurrence in Southern China (odds ratio 1.999 [95% CI 1.229-3.251], P = 0.005). By using transmitochondrial technology, we demonstrated that the activity of respiratory chain complexes was lower in the case of mtDNA haplogroup N9a (N9a1 and N9a10a) than in three non-N9a haplogroups (D4j, G3a2, and Y1) and that this could lead to alterations in mitochondrial function and mitochondrial redox status. Transcriptome analysis revealed that OXPHOS function and metabolic regulation differed markedly between N9a and non-N9a cybrids. Furthermore, in N9a cybrids, insulin-stimulated glucose uptake might be inhibited at least partially through enhanced stimulation of ERK1/2 phosphorylation and subsequent TLR4 activation, which was found to be mediated by the elevated redox status in N9a cybrids. Although it remains unclear whether other signaling pathways (e.g., Wnt pathway) contribute to the T2D susceptibility of haplogroup N9a, our data indicate that in the case of mtDNA haplogroup N9a, T2D is affected, at least partially through ERK1/2 overstimulation and subsequent TLR4 activation.