Function of GRIM-19, a Mitochondrial Respiratory Chain Complex I Protein, in Innate Immunity

Mitochondria respiratory chain (RC), consisting of five multisubunit complexes, is crucial for cellular energy production, reactive oxygen species generation, and regulation of apoptosis. Recently, a few mitochondrial proteins have been reported to be essential for innate immunity, but the function of mitochondrial RC in innate immunity is largely unknown. By knock-out of GRIM-19, a newly identified subunit protein of mitochondrial complex I, in mice, we found that heterogeneous mice (GRIM-19(+/-)) are prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus. Macrophages derived from these mice have compromised mitochondrial complex I activity and increased reactive oxygen species level. Bacterial infection induces a rapid up-regulation of GRIM-19 and complex I activity in the wild-type macrophages, but both are reduced in the macrophages from GRIM-19(+/-) mice. These cells also have decreased intracellular killing ability against S. saprophyticus. The defects for this probably occur in the fusion of bacteria to lysosome, but not in the bacterial engulfment and macrophage migration. In addition, production of proinflammatory cytokines, such as interleukin (IL)-1, IL-12, IL-6, and interferon (IFN)-γ, induced by both bacterial infection and lipopolysaccharide (LPS) and monodansylcadaverine treatment, is also decreased in the GRIM19(+/-) macrophages. Inhibition of mitochondrial RC activity by inhibitors shows a similar reduction on the cytokine production. Due to low cytokine production, the inflammatory response caused by in vivo bacterial challenge in the bladders of GRIM-19(+/-) mice is compromised. This study provides genetic evidence for a critical role of mitochondrial RC in innate immunity.     

GRIM-19 is essential for maintenance of mitochondrial membrane potential

GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain and subsequently was demonstrated to be involved in complex I assembly and activity. To further understand its function in complex I, we dissected its functional domains by generating a number of deletion, truncation, and point mutants. The mitochondrial localization sequences were located at the N-terminus. Strikingly, deletion of residues 70-80, 90-100, or the whole C-terminal region (70-144) led to a loss of mitochondrial transmembrane potential (DeltaPsim). However, similar deletions of another two complex I subunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletion of the last 10 residues affected GRIM-19's ability to be assembled to complex I. We constructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintain normal DeltaPsim. Cells overexpressing this mutant did not spontaneously undergo cell death, but were sensitized to apoptosis induced by cell death agents. Our results demonstrate that GRIM-19 is required for electron transfer activity of complex I, and disruption of DeltaPsim by GRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.     

The cell death regulator GRIM-19 is involved in HIV-1 induced T-cell apoptosis

One of the hallmarks of Human Immunodeficiency Virus-1 (HIV-1) infection is progressive depletion of the infected and bystander CD4+ T-cells by apoptosis. Different mitochondrial proteins have been implicated in this apoptotic process; however, the role of different subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes in apoptosis is not clearly understood. Some of the OXPHOS complex subunits seem to perform other functions in addition to their primary role in energy generating process. GRIM-19 (gene associated with retinoid-interferon-induced-mortality-19), a subunit of mitochondrial complex-I was previously implicated in Interferon-β and retionoic acid induced apoptosis in many tumor cells. In this study we report, using differential gene expression analysis, that GRIM-19 is up-regulated in HIV-1 infected apoptotic T-cells. A temporal up regulation of this subunit was observed in different HIV-1 infected T-cell lines and human PBMC and the extent of increase correlated to increasing apoptosis and virus production. Moreover, silencing GRIM-19 in HIV-1 infected cells reduced apoptosis, indicating its involvement in HIV-1 induced T-cell death.     

Expression and functional characterization of a gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) from orange-spotted grouper (Epinephelus coioides)

GRIM-19 is a nuclear encoded subunit of complex I that has been implicated in apoptosis. The protein participates in multiple functions including the innate immune response. GRIM-19 has been studied in humans and other mammals; however, fish GRIM-19 has not been well characterized. In this study, a new GRIM-19 gene, EcGRIM-19, was isolated from the orange-spotted grouper (Epinephelus coioides) cDNA library, which was constructed following LPS treatment. EcGRIM-19 is a 582-bp gene that encodes a 144-amino acid protein. The gene is a true ortholog of mammalian GRIM-19. EcGRIM-19 exhibits ubiquitous and constitutive expression in the different tissues of the orange-spotted grouper. The expression levels of EcGRIM-19 are altered in the gill, spleen, kidney and liver after induction with LPS. The subcellular localization analysis demonstrated that the EcGRIM-19 protein is localized predominantly in the mitochondria. In addition, amino acids 30–50 of the protein are responsible for the mitochondrial localization of EcGRIM-19. The caspase assay demonstrated that the overexpression of GRIM-19 enhanced the cellular sensitivity to interferon(IFN)-β- and retinoic acid (RA)-induced death in HeLa cells. The data presented in this study are important for further understanding the EcGRIM-19 gene function in fish.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

Suppression of Mitochondrial Complex I Influences Cell Metastatic Properties

Despite the fact that mitochondrial dysfunction has an important role in tumorigenesis and metastasis, the underlying mechanism remains to be elucidated. Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) is the first and the largest protein complex of the mitochondrial electron-transport chain (ETC),which has an essential role in maintaining mitochondrial function and integrity. In this study, we separately knocked down two subunits of mitochondrial complex I, GRIM-19 or NDUFS3, and investigated their effects on metastatic behaviors and explored the possible mechanisms. Our data showed that stable down-modulation of GRIM-19 or NDUFS3 decreased complex I activity and reactive oxygen species (ROS) production; led to enhanced cell adhesion, migration, invasion, and spheroid formation; and influenced the expressions of extracellular matrix (ECM) molecules and its related proteins. We also observed that the expressions of GRIM-19, NDUFS3, and ECM elements were correlated with invasive capabilities of breast cancer cell lines. These results suggest that inhibition of complex I affects metastatic properties of cancer cells, and mitochondrial ROS might play a crucial role in these processes by regulating ECM.     

c-Src kinase impairs the expression of mitochondrial OXPHOS complexes in liver cancer

Src family kinases (SFKs) play a crucial role in the regulation of multiple cellular pathways, including mitochondrial oxidative phosphorylation (OXPHOS). Aberrant activities of one of the most predominant SFKs, c-Src, was identified as a fundamental cause for dysfunctional cell signaling and implicated in cancer development and metastasis, especially in human hepatocellular carcinoma (HCC). Recent work in our laboratory revealed that c-Src is implicated in the regulation of mitochondrial energy metabolism in cancer. In this study, we investigated the effect of c-Src expression on mitochondrial energy metabolism by examining changes in the expression and activities of OXPHOS complexes in liver cancer biopsies and cell lines. An increased expression of c-Src was correlated with an impaired expression of nuclear- and mitochondrial-encoded subunits of OXPHOS complexes I and IV, respectively, in metastatic biopsies and cell lines. Additionally, we observed a similar association between high c-Src and reduced OXPHOS complex expression and activity in mouse embryonic fibroblast (MEF) cell lines. Interestingly, the inhibition of c-Src kinase activity with the SFK inhibitor PP2 and c-Src siRNA stimulated the expression of complex I and IV subunits and increased their enzymatic activities in both cancer and normal cells. Evidence provided in this study reveals that c-Src impairs the expression and function of mitochondrial OXPHOS complexes, resulting in a significant defect in mitochondrial energy metabolism, which can be a contributing factor to the development and progression of liver cancer. Furthermore, our findings strongly suggest that SFK inhibitors should be used in the treatment of HCC and other cancers with aberrant c-Src kinase activity to improve mitochondrial energy metabolism.     

Initiation of Electron Transport Chain Activity in the Embryonic Heart Coincides with the Activation of Mitochondrial Complex 1 and the Formation of Supercomplexes

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.     

Molecular base of biochemical complex I deficiency

The oxidative phosphorylation (OXPHOS) system, consisting of five enzyme complexes (I-V) together with 2 electron carriers, has an important role in the energy metabolism of the cell. With 45 subunits, complex I is the first and largest complex of the respiratory chain. It is under bigenomic control and a proper interaction between the mitochondrial and the nuclear genome is important for a good biogenesis and functioning of the complex. Isolated complex I deficiency is the most frequently diagnosed form of mitochondrial disorders caused by the disturbance of the OXPHOS system. It has a wide clinical variety and, at present, in many patients the underlying genetic cause of the complex I deficiency is still not known. In this review, the role of complex I in the oxidative phosphorylation and the localization and function of the different complex I subunits will be described. Furthermore, a brief overview of the assembly process and biochemical studies, performed when a patient is suspected of a mitochondrial disorder is given. Finally, the present knowledge for molecular base of complex I deficiency is described and the findings in a research cohort of patients with complex I deficiency are reported. Identifying new genes encoding proteins involved in complex I biogenesis is challenging and in the near future new powerful techniques will make high throughput screening possible. Progress in elucidating the genetic defect causing complex I deficiencies is important for a better genetic counseling, prenatal diagnostic possibilities and further development of new treatment strategies to cure the complex I deficiencies in the future.     

Enhanced liver but not muscle OXPHOS in diabetes and reduced glucose output by complex I inhibition

Mitochondrial function is critical in energy metabolism. To fully capture how the mitochondrial function changes in metabolic disorders, we investigated mitochondrial function in liver and muscle of animal models mimicking different types and stages of diabetes. Type 1 diabetic mice were induced by streptozotocin (STZ) injection. The db/db mice were used as type 2 diabetic model. High-fat diet-induced obese mice represented pre-diabetic stage of type 2 diabetes. Oxidative phosphorylation (OXPHOS) of isolated mitochondria was measured with Clark-type oxygen electrode. Both in early and late stages of type 1 diabetes, liver mitochondrial OXPHOS increased markedly with complex IV-dependent OXPHOS being the most prominent. However, ATP, ADP and AMP contents in the tissue did not change. In pre-diabetes and early stage of type 2 diabetes, liver mitochondrial complex I and II-dependent OXPHOS increased greatly then declined to almost normal at late stage of type 2 diabetes, among which alteration of complex I-dependent OXPHOS was the most significant. In contrast, muscle mitochondrial OXPHOS in HFD, early-stage type 1 and 2 diabetic mice, did not change. In vitro, among inhibitors to each complex, only complex I inhibitor rotenone decreased glucose output in primary hepatocytes without cytotoxicity both in the absence and presence of oleic acid (OA). Rotenone affected cellular energy state and had no effects on cellular and mitochondrial reactive oxygen species production. Taken together, the mitochondrial OXPHOS of liver but not muscle increased in obesity and diabetes, and only complex I inhibition may ameliorate hyperglycaemia via lowering hepatic glucose production.     

Ribonucleoprotein Y-box-binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homoeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1α subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1β subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.     

The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes

The organization of the oxidative phosphorylation (OXPHOS) system within the inner mitochondrial membrane appears to be far more complicated than previously thought. In particular, the individual protein complexes of the OXPHOS system (complexes I to V) were found to specifically interact forming defined supramolecular structures. Blue-native polyacrylamide gel electrophoresis and single particle electron microscopy proved to be especially valuable in studying the so-called "respiratory supercomplexes". Based on these procedures, increasing evidence was presented supporting a "solid state" organization of the OXPHOS system. Here, we summarize results on the formation, organisation and function of the various types of mitochondrial OXPHOS supercomplexes.     

Altered mitochondrial functioning induced by preoperative fasting may underlie protection against renal ischemia/reperfusion injury

We reported previously that the robust protection against renal ischemia/reperfusion (I/R) injury in mice by fasting was largely initiated before the induction of renal I/R. In addition, we found that preoperative fasting downregulated the gene expression levels of complexes I, IV, and V of the mitochondrial oxidative phosphorylation (OXPHOS) system, while it did not change those of complexes II and III. Hence, we now investigated the effect of 3 days of fasting on the functioning of renal mitochondria in order to better understand our previous findings. Fasting did not affect mitochondrial density. Surprisingly, fasting significantly increased the protein expression of complex II of the mitochondrial OXPHOS system by 19%. Complex II‐driven state 3 respiratory activity was significantly reduced by fasting (46%), which could be partially attributed to the significant decrease in the enzyme activity of complex II (16%). Fasting significantly inhibited Ca²⁺‐dependent mitochondrial permeability transition pore opening that is directly linked to protection against renal I/R injury. The inhibition of the mitochondrial permeability transition pore did not involve the expression of the voltage‐dependent anion channel by fasting. In conclusion, 3 days of fasting clearly induces the inhibition of complex II‐driven mitochondrial respiration state 3 in part by decreasing the amount of functional complex II, and inhibits mitochondrial permeability transition pore opening. This might be a relevant sequence of events that could contribute to the protection of the kidney against I/R injury. J. Cell. Biochem. 114: 230–237, 2012. © 2012 Wiley Periodicals, Inc.     

Human mitochondrial complex I assembly: a dynamic and versatile process

One can but admire the intricate way in which biomolecular structures are formed and cooperate to allow proper cellular function. A prominent example of such intricacy is the assembly of the five inner membrane embedded enzymatic complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system, which involves the stepwise combination of >80 subunits and prosthetic groups encoded by both the mitochondrial and nuclear genomes. This review will focus on the assembly of the most complicated OXPHOS structure: complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Recent studies into complex I assembly in human cells have resulted in several models elucidating a thus far enigmatic process. In this review, special attention will be given to the overlap between the various assembly models proposed in different organisms. Complex I being a complicated structure, its assembly must be prone to some form of coordination. This is where chaperone proteins come into play, some of which may relate complex I assembly to processes such as apoptosis and even immunity.     

Association of Genes,Pathways, and Haplogroups of the Mitochondrial Genome with the Risk of Colorectal Cancer: The Multiethnic Cohort

The mitochondrial genome encodes for the synthesis of 13 proteins that are essential for the oxidative phosphorylation (OXPHOS) system. Inherited variation in mitochondrial genes may influence cancer development through changes in mitochondrial proteins, altering the OXPHOS process, and promoting the production of reactive oxidative species. To investigate the role of the OXPHOS pathway and mitochondrial genes in colorectal cancer (CRC) risk, we tested 185 mitochondrial SNPs (mtSNPs), located in 13 genes that comprise four complexes of the OXPHOS pathway and mtSNP groupings for rRNA and tRNA, in 2,453 colorectal cancer cases and 11,930 controls from the Multiethnic Cohort Study. Using the sequence kernel association test, we examined the collective set of 185 mtSNPs, as well as subsets of mtSNPs grouped by mitochondrial pathways, complexes, and genes, adjusting for age, sex, principal components of global ancestry, and self-reported maternal race/ethnicity. We also tested for haplogroup associations using unconditional logistic regression, adjusting for the same covariates. Stratified analyses were conducted by self-reported maternal race/ethnicity. In European Americans, a global test of all genetic variants of the mitochondrial genome identified an association with CRC risk (P = 0.04). In mtSNP-subset analysis, the NADH dehydrogenase 2 (MT-ND2) gene in Complex I was associated with CRC risk at a P-value of 0.001 (q = 0.015). In addition, haplogroup T was associated with CRC risk (OR = 1.66, 95% CI: 1.19–2.33, P = 0.003). No significant mitochondrial pathway and gene associations were observed in the remaining four racial/ethnic groups—African Americans, Asian Americans, Latinos, and Native Hawaiians. In summary, our findings suggest that variations in the mitochondrial genome and particularly in the MT-ND2 gene may play a role in CRC risk among European Americans, but not in other maternal racial/ethnic groups. Further replication is warranted and future studies should evaluate the contribution of mitochondrial proteins encoded by both the nuclear and mitochondrial genomes to CRC risk.     

GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I)

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Ilambda, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-beta and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.     

In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis

Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.KEY WORDS: Mitochondrial respiratory chain, Nonalcoholic steatohepatitis, NADPH oxidase, Oxidative phosphorylation, Proteomic, Nitro-oxidative stress, OXPHOS