Mitochondrial Solute Carrier Research Articles

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.

Unusual structure and splicing pattern of the vertebrate mitochondrial solute carrier SLC25A3 gene

The DNA sequence corresponding to the second exon of the SLC25A3 gene is duplicated in vertebrates. The second exon codes for the first transmembrane segment and parts of the immediately adjoining intermembrane and mitochondrial matrix segments. The two genomic exon 2 sequences are 84% similar in zebrafish (slc25a3b gene), 70% in chicken, 66% in mouse and 67% in human. The amino acid identity is 86% in zebrafish, 77% in chicken and 70% in mouse and human. The two copies of exon 2 are separated by an intronic interval. Translation of both exon 2 sequences would alter the reading frame of the downstream sequence, generating a modified aa sequence which would soon be truncated by a stop codon. As a matter of fact the splicing machinery is tuned in such a way that in some species only one of the two copies is expressed and the other is spliced out, while in other species both copies are expressed but only one at a time, generating two alternative protein products.

Decreased expression of the mitochondrial solute carrier SLC25A43 in basal cell carcinoma compared with healthy skin.

Basal cell carcinoma is the most common type of cancer in fair-skinned individuals, and its incidence is rapidly increasing. The aim of the present study was to investigate the gene and protein expression of the mitochondrial solute carrier family 25 member 43 (SLC25A43) in basal cell carcinoma. SLC25A43 has previously been identified to be genetically altered and associated with cell proliferation in human epidermal growth factor receptor 2-positive breast cancer. However, the knowledge about SLC25A43 is limited, and its role in other cancers is unknown. The SLC25A43 gene and protein expression was analysed in 14 basal cell carcinomas and healthy skin samples from the same individuals by quantitative polymerase chain reaction and immunohistochemistry, respectively. The results demonstrated a significantly lower (≥50%) SLC25A43 gene expression in all carcinomas compared with that in healthy skin. In addition, SLC25A43 protein expression was absent in >90% of all visual fields in the basal cell carcinomas, and the H-score was significantly lower in tumours compared with the adjacent epidermis. These results demonstrate that SLC25A43 expression is altered at the gene and protein levels in basal cell carcinoma. The underlying mechanisms and the clinical relevance of these data must be elucidated in additional experimental and clinical studies.

Conservation/Mutation in the Splice Sites of Mitochondrial Solute Carrier Genes of Vertebrates.

The "canonical" introns begin by the dinucleotide GT and end by the dinucleotide AG. GT, together with a few downstream nucleotides, and AG, with a few of the immediately preceding nucleotides, are thought to be the strongest splicing signals (5'ss and 3'ss, respectively). We examined the composition of the intronic initial and terminal hexanucleotides of the mitochondrial solute carrier genes (SLC25A's) of zebrafish, chicken, mouse, and human. These genes are orthologous and we selected the transcripts in which the arrangement of exons and introns was superimposable in the species considered. Both 5'ss and 3'ss were highly polymorphic, with 104 and 126 different configurations, respectively, in our sample. In the line of evolution from zebrafish to chicken, as well as in that from zebrafish to mammals, the average nucleotide conservation in the four variable nucleotides was about 50% at 5' and 40% at 3'. In the divergent evolution of mouse and human, the conservation was about 80% at 5' and 70% at 3'. Despite these changes, the splicing signals remain strong enough to operate at the same site. At both 5' and 3', the frequency of a nucleotide at a given position in the zebrafish sequence is positively correlated with its conservation in chicken and mammals, suggesting that selection continued to operate in birds and mammals along similar lines.

Abstract PR07: Functional identification of a mitochondrial glutamine carrier using isotope tracers

Abstract Mitochondria are the central metabolic hub of cells, and cells have the ability to metabolize numerous substrates in biochemical pathways within these organelles. Glutamine is a vital nutrient that serves as both nitrogen carrier and anaperotic substrate for mammalian cells. Various cancer cells exhibit dependence on glutaminolysis to fuel bioenergetic and biosynthetic metabolic pathways. This pathway is catalyzed, in part, via mitochondrial glutaminase; however, the mechanism through which glutamine is transported into the matrix is not well known. In fact, the function of nearly half the known mitochondrial solute carriers (SLC25 family) is yet to be described. Using a combination of cell engineering strategies, metabolic tracing using 15N and 13C labeled substrates, and respirometry applied to both intact and permeabilized cultures we have identified a putative mitochondrial glutamine carrier (MQC). Knockdown of this carrier decreases cell growth and drives cells to use alternate pathways for oxidation of glutamine in the TCA cycle. Cancer cells treated with inhibitors of glutaminase exhibit a similar metabolic phenotype as demonstrated via 13C tracing. These results functionally identify a critical node in mitochondrial metabolism and further highlight the utility of metabolic tracing and mass spectrometry for elucidating metabolic function. Citation Format: Christian Metallo. Functional identification of a mitochondrial glutamine carrier using isotope tracers. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR07.

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2.

Asymmetric dimethyl L-arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the carrier protein(s) that transport ADMA across the inner mitochondrial membrane. It was found that the recombinant, purified mitochondrial solute carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38±0.04mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function.

Identification of the Vertebrate RIM2 Ortholog by Genetic Complementation for Heme

Heme is the critical cofactor in hemoglobin responsible for its oxygen‐carrying properties. Mfrn1 (SLC25A37), functions as the major iron importer into the mitochondria for heme synthesis. The function of Mfrn1 is highly conserved from vertebrates to yeasts given the universal requirement for heme in many biological processes. The over‐expression of a related mitochondrial solute carrier, RIM2, allowed yeast deficient for the Mfrn1 orthologs, MRS3/4, to grow on low‐iron media, thereby implicating a role for other iron transporters in heme synthesis.HypothesisTwo potential RIM2 vertebrate orthologs, SLC25A33 and SLC25A36, play a role in mitochondrial iron import to complement the hemoglobinization‐defect of Mfrn1‐deficiency in a zebrafish model.Using protein homology search, two top candidates, SLC25A33 and SLC25A36, were identified as potential orthologs for yeast RIM2. Others recently showed that these 2 carriers complemented the pyrimidine‐deficiency phenotype of ΔRIM2 yeasts (Di Noia 2014 JBC); although, the iron‐auxotrophy was not investigated.Specific Aim 1The over‐expression of vertebrate RIM2 orthologs will be evaluated for ability to rescue the anemia in Mfrn1‐deficient zebrafish, frascati.We will examine whether the over‐expression of the vertebrate RIM2 orthologs could supply sufficient iron to enable mitochondrial heme synthesis in absence of Mfrn1, thereby correcting the hemoglobin‐defect. For this purpose, we will microinject cRNA with either SLC25A33 or SLC25A36 into frascati zygotes and evaluate the restoration of hemoglobinized erythrocytes.AcknowledgmentsThis work was supported by grants from the APS (JMC, AN) and the NIH (ABD, BHP).

Determinism and randomness in the evolution of introns and sine inserts in mouse and human mitochondrial solute carrier and cytokine receptor genes

In the homologous genes studied, the exons and introns alternated in the same order in mouse and human. We studied, in both species: corresponding short segments of introns, whole corresponding introns and complete homologous genes. We considered the total number of nucleotides and the number and orientation of the SINE inserts. Comparisons of mouse and human data series showed that at the level of individual relatively short segments of intronic sequences the stochastic variability prevails in the local structuring, but at higher levels of organization a deterministic component emerges, conserved in mouse and human during the divergent evolution, despite the ample re-editing of the intronic sequences and the fact that processes such as SINE spread had taken place in an independent way in the two species. Intron conservation is negatively correlated with the SINE occupancy, suggesting that virus inserts interfere with the conservation of the sequences inherited from the common ancestor.

OPA1 and mitochondrial solute carriers in bioenergetic metabolism

The mitochondrial fusion protein optic atrophy 1 (OPA1) is required to maintain cristae structure and for ATP synthase assembly. Our recent work demonstrates that OPA1 dynamically regulates these processes by sensing changes in nutrient availability through mitochondrial solute carriers and adjusting the metabolic output of mitochondria accordingly. This is a critical survival process as its inhibition leads to cell death.

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.

Overexpression of SLC25A38 protein on acute lymphoblastic leukemia cells

SLC25A38 is a recently identified protein that belongs to the mitochondrial solute carrier family, SLC25. Previous studies have shown that it is a pro-apoptotic protein, which regulates intrinsic caspase-dependent apoptosis. In order to clarify the effect of SLC25A38 protein expression on acute lymphoblastic leukemia (ALL) cells, we detected the expression of SLC25A38 in various cell lines (RPMI 8226, U266, Molt-4 and Jurkat) by western blot analysis. The results indicate that SLC25A38 is highly expressed in the four cell lines. Among 55 leukemia patients (adult, n=32 and infant, n=23), a high expression of SLC25A38 protein was observed in seven infant (7/23, 30.4%) and 15 adult (15/32, 46.9%) ALL patients. Two adult ALL patients that were positive for SLC25A38 were analyzed and the level of SLC25A38 significantly reduced or disappeared following combined chemotherapy, however, reappeared upon ALL recurrence. The expression level was identified to be associated with the proportion of blast cells in the bone marrow. Additionally, SLC25A38 and Notch1 were co-expressed in the four cell lines and the ALL patient samples. The present results show that expression of SLC25A38 is a common feature of ALL cells and may be a novel biomarker for diagnosis, as well as a potential therapeutic target for ALL.

The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.

Expression of genes involved in fatty acid transport and insulin signaling is altered by physical inactivity and exercise training in human skeletal muscle

Physical deconditioning is associated with the development of chronic diseases, including type 2 diabetes and cardiovascular disease. Exercise training effectively counteracts these developments, but the underlying mechanisms are largely unknown. To gain more insight into these mechanisms, muscular gene expression levels were assessed after physical deconditioning and after exercise training of the lower limbs in humans by use of gene expression microarrays. To exclude systemic effects, we used human models for local physical inactivity (3 wk of unilateral limb suspension) and for local exercise training (6 wk of functional electrical stimulation exercise of the extremely deconditioned legs of individuals with a spinal cord injury). The most interesting subset of genes, those downregulated after deconditioning as well as upregulated after exercise training, contained 18 genes related to both the "insulin action" and "adipocytokine signaling" pathway. Of these genes, the three with strongest up/downregulation were the muscular fatty acid-binding protein-3 (FABP3), the fatty acid oxidizing enzyme hydroxyacyl-CoA dehydrogenase (HADH), and the mitochondrial fatty acid transporter solute carrier 25 family member A20 (SLC25A20). The expression levels of these genes were confirmed using RT-qPCR. The results of the present study indicate an important role for a decreased transport and metabolism of fatty acids, which provides a link between physical activity levels and insulin signaling.

Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction

Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I–V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed “mild uncoupling.” UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed.

Reduction of Mitoferrin Results in Abnormal Development and Extended Lifespan in Caenorhabditis elegans

Iron is essential for organisms. It is mainly utilized in mitochondria for biosynthesis of iron-sulfur clusters, hemes and other cofactors. Mitoferrin 1 and mitoferrin 2, two homologues proteins belonging to the mitochondrial solute carrier family, are required for iron delivery into mitochondria. Mitoferrin 1 is highly expressed in developing erythrocytes which consume a large amount of iron during hemoglobinization. Mitoferrin 2 is ubiquitously expressed, whose functions are less known. Zebrafish with mitoferrin 1 mutation show profound hypochromic anaemia and erythroid maturation arrests, and yeast with defects in MRS3/4, the counterparts of mitoferrin 1/2, has low mitochondrial iron levels and grows poorly by iron depletion. Mitoferrin 1 expression is up-regulated in yeast and mouse models of Fiedreich's ataxia disease and in human cell culture models of Parkinson disease, suggesting its involvement in the pathogenesis of diseases with mitochondrial iron accumulation. In this study we found that reduced mitoferrin levels in C. elegans by RNAi treatment causes pleiotropic phenotypes such as small body size, reduced fecundity, slow movement and increased sensitivity to paraquat. Despite these abnormities, lifespan was increased by 50% to 80% in N2 wild type strain, and in further studies using the RNAi sensitive strain eri-1, more than doubled lifespan was observed. The pathways or mechanisms responsible for the lifespan extension and other phenotypes of mitoferrin RNAi worms are worth further study, which may contribute to our understanding of aging mechanisms and the pathogenesis of iron disorder related diseases.

Identifying an APP-binding protein in neuronal cell death

Results Here we find that a mitochondrial solute carrier family protein, appoptosin, induces reactive oxygen species release and intrinsic caspase-dependent apoptosis. The physiological function of appoptosin is to transport/ exchange glycine/5-amino-levulinic acid across the mitochondrial membrane for heme synthesis. Alzheimer’s b-amyloid precursor protein interacts with appoptosin and modulates appoptosin-induced apoptosis. Levels of appoptosin are upregulated in brain samples from Alzheimer’s disease and infarct patients and in rodent stroke models, as well as in cells treated with b-amyloid (Ab) and glutamate. Downregulation of appoptosin prevents the cell death and caspase activation caused by glutamate or Ab insults.

Biophysical Properties of UCP1

Brown adipose tissue (BAT) is a specialized mammalian organ that coverts body fat reserves into heat. This conversion is mediated by BAT mitochondria and is important for maintaining body temperature and reducing fat depositions. Burning fat in BAT mitochondria does not result in ATP synthesis due to unusually high conductance of the inner mitochondrial membrane (IMM). This BAT-specific conductance dissipates the mitochondrial electrochemical gradient used in other tissues for ATP production and converts the energy of fatty acid oxidation into heat. The molecule responsible for the high conductance of the IMM and uncoupling of oxidative phosphorylation in BAT mitochondria is uncoupling protein 1 (UCP1). UCP1 is a BAT-specific protein that belongs to the superfamily of mitochondrial solute carriers (SLC25). UCP1-mediated uncoupling is activated by fatty acids and inhibited by purine nucleotides. In spite of the fact that UCP1 was identified decades ago, the mechanism of UCP1 operation is unknown due to lack of direct methods to study its activity. Here, we resolve the problem by directly recording UCP1 currents across the IMM of BAT mitochondria using the patch-clamp technique. To identify UCP1 current, we compare currents recorded from the wild-type and UCP1 (-/-) mitochondria. The detailed electrophysiological analysis demonstrated that UCP1 translocates fatty acid anion in symport with H+, while the fatty acid hydrophobic tail prevents dissociation of fatty acid from UCP1, causing UCP1 to operate as H+ carrier. This work establishes the mechanism of fatty acid dependent mitochondrial uncoupling which is implicated in metabolic and age-related diseases.

Identification of a Redox-Modulatory Interaction Between Uncoupling Protein 3 and Thioredoxin 2 in the Mitochondrial Intermembrane Space

Uncoupling protein 3 (UCP3) is a member of the mitochondrial solute carrier superfamily that is enriched in skeletal muscle and controls mitochondrial reactive oxygen species (ROS) production, but the mechanisms underlying this function are unclear. The goal of this work focused on the identification of mechanisms underlying UCP3 functions. Here we report that the N-terminal, intermembrane space (IMS)-localized hydrophilic domain of mouse UCP3 interacts with the N-terminal mitochondrial targeting signal of thioredoxin 2 (Trx2), a mitochondrial thiol reductase. Cellular immunoprecipitation and in vitro pull-down assays show that the UCP3-Trx2 complex forms directly, and that the Trx2 N-terminus is both necessary and sufficient to confer UCP3 binding. Mutation studies show that neither a catalytically inactivated Trx2 mutant, nor a mutant Trx2 bearing the N-terminal targeting sequence of cytochrome c oxidase (COXMTS-Trx2) bind UCP3. Biochemical analyses using permeabilized mitochondria, and live cell experiments using bimolecular fluorescence complementation show that the UCP3-Trx2 complex forms specifically in the IMS. Finally, studies in C2C12 myocytes stably overexpressing UCP3 (2.5-fold) and subjected to Trx2 knockdown show that Trx2 is required for the UCP3-dependent mitigation of complex III-driven mitochondrial ROS generation. UCP3 expression was increased in mice fed a high fat diet, leading to increased localization of Trx2 to the IMS. UCP3 overexpression also increased expression of the glucose transporter GLUT4 in a Trx2-dependent fashion. This is the first report of a mitochondrial protein-protein interaction with UCP3 and the first demonstration that UCP3 binds directly, and in cells and tissues with mitochondrial thioredoxin 2. These studies identify a novel UCP3-Trx2 complex, a novel submitochondrial localization of Trx2, and a mechanism underlying UCP3-regulated mitochondrial ROS production.

MISC-1/OGC links mitochondrial metabolism, apoptosis and insulin secretion.

We identified MISC-1 (Mitochondrial Solute Carrier) as the C. elegans orthologue of mammalian OGC (2-oxoglutarate carrier). OGC was originally identified for its ability to transfer α-ketoglutarate across the inner mitochondrial membrane. However, we found that MISC-1 and OGC are not solely involved in metabolic control. Our data show that these orthologous proteins participate in phylogenetically conserved cellular processes, like control of mitochondrial morphology and induction of apoptosis. We show that MISC-1/OGC is required for proper mitochondrial fusion and fission events in both C. elegans and human cells. Transmission electron microscopy reveals that loss of MISC-1 results in a decreased number of mitochondrial cristae, which have a blebbed appearance. Furthermore, our pull-down experiments show that MISC-1 and OGC interact with the anti-apoptotic proteins CED-9 and Bcl-xL, respectively, and with the pro-apoptotic protein ANT. Knock-down of misc-1 in C. elegans and OGC in mouse cells induces apoptosis through the caspase cascade. Genetic analysis suggests that MISC-1 controls apoptosis through the physiological pathway mediated by the LIN-35/Rb-like protein. We provide genetic and molecular evidence that absence of MISC-1 increases insulin secretion and enhances germline stem cell proliferation in C. elegans. Our study suggests that the mitochondrial metabolic protein MISC-1/OGC integrates metabolic, apoptotic and insulin secretion functions. We propose a novel mechanism by which mitochondria integrate metabolic and cell survival signals. Our data suggest that MISC-1/OGC functions by sensing the metabolic status of mitochondria and directly activate the apoptotic program when required. Our results suggest that controlling MISC-1/OGC function allows regulation of mitochondrial morphology and cell survival decisions by the metabolic needs of the cell.

A20 Modulates Lipid Metabolism and Energy Production to Promote Liver Regeneration

BackgroundLiver Regeneration is clinically of major importance in the setting of liver injury, resection or transplantation. We have demonstrated that the NF-κB inhibitory protein A20 significantly improves recovery of liver function and mass following extended liver resection (LR) in mice. In this study, we explored the Systems Biology modulated by A20 following extended LR in mice.Methodology and Principal FindingsWe performed transcriptional profiling using Affymetrix-Mouse 430.2 arrays on liver mRNA retrieved from recombinant adenovirus A20 (rAd.A20) and rAd.βgalactosidase treated livers, before and 24 hours after 78% LR. A20 overexpression impacted 1595 genes that were enriched for biological processes related to inflammatory and immune responses, cellular proliferation, energy production, oxidoreductase activity, and lipid and fatty acid metabolism. These pathways were modulated by A20 in a manner that favored decreased inflammation, heightened proliferation, and optimized metabolic control and energy production. Promoter analysis identified several transcriptional factors that implemented the effects of A20, including NF-κB, CEBPA, OCT-1, OCT-4 and EGR1. Interactive scale-free network analysis captured the key genes that delivered the specific functions of A20. Most of these genes were affected at basal level and after resection. We validated a number of A20's target genes by real-time PCR, including p21, the mitochondrial solute carriers SLC25a10 and SLC25a13, and the fatty acid metabolism regulator, peroxisome proliferator activated receptor alpha. This resulted in greater energy production in A20-expressing livers following LR, as demonstrated by increased enzymatic activity of cytochrome c oxidase, or mitochondrial complex IV.ConclusionThis Systems Biology-based analysis unravels novel mechanisms supporting the pro-regenerative function of A20 in the liver, by optimizing energy production through improved lipid/fatty acid metabolism, and down-regulated inflammation. These findings support pursuit of A20-based therapies to improve patients’ outcomes in the context of extreme liver injury and extensive LR for tumor treatment or donation.