Baf155 regulates skeletal muscle metabolism via HIF-1a signaling.

During exercise, skeletal muscle is exposed to a low oxygen condition, hypoxia. Under hypoxia, the transcription factor hypoxia-inducible factor-1α (HIF-1α) is stabilized and induces expressions of its target genes regulating glycolytic metabolism. Here, using a skeletal muscle-specific gene ablation mouse model, we show that Brg1/Brm-associated factor 155 (Baf155), a core subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, is essential for HIF-1α signaling in skeletal muscle. Muscle-specific ablation of Baf155 increases oxidative metabolism by reducing HIF-1α function, which accompanies the decreased lactate production during exercise. Furthermore, the augmented oxidation leads to high intramuscular adenosine triphosphate (ATP) level and results in the enhancement of endurance exercise capacity. Mechanistically, our chromatin immunoprecipitation (ChIP) analysis reveals that Baf155 modulates DNA-binding activity of HIF-1α to the promoters of its target genes. In addition, for this regulatory function, Baf155 requires a phospho-signal transducer and activator of transcription 3 (pSTAT3), which forms a coactivator complex with HIF-1α, to activate HIF-1α signaling. Our findings reveal the crucial role of Baf155 in energy metabolism of skeletal muscle and the interaction between Baf155 and hypoxia signaling.

Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K.

How body size is determined is a long-standing question in biology, yet its regulatory mechanisms remain largely unknown. Here, we find that a conserved microRNA miR-8 and its target, USH, regulate body size in Drosophila. miR-8 null flies are smaller in size and defective in insulin signaling in fat body that is the fly counterpart of liver and adipose tissue. Fat body-specific expression and clonal analyses reveal that miR-8 activates PI3K, thereby promoting fat cell growth cell-autonomously and enhancing organismal growth non-cell-autonomously. Comparative analyses identify USH and its human homolog, FOG2, as the targets of fly miR-8 and human miR-200, respectively. USH/FOG2 inhibits PI3K activity, suppressing cell growth in both flies and humans. FOG2 directly binds to p85alpha, the regulatory subunit of PI3K, and interferes with the formation of a PI3K complex. Our study identifies two novel regulators of insulin signaling, miR-8/miR-200 and USH/FOG2, and suggests their roles in adolescent growth, aging, and cancer.

Reduced branched-chain aminotransferase activity alleviates metabolic vulnerability caused by dim light exposure at night in Drosophila.

The rhythmic pattern of biological processes controlled by light over 24 h is termed the circadian rhythm. Disturbance of circadian rhythm due to exposure to light at night (LAN) disrupts the sleep-wake cycle and can promote cardiovascular disease, diabetes, cancer, and metabolic disorders in humans. We studied how dim LAN affects the circadian rhythm and metabolism using male Drosophila. Wild-type flies exposed to the dim light of 10 lux at night displayed altered 24 h sleep-wake behavior and expression patterns of circadian rhythm genes. In addition, the flies became more vulnerable to metabolic stress, such as starvation. Whole-body metabolite analysis revealed decreased amounts of branched-chain amino acids (BCAAs), such as isoleucine and valine. The dim light exposure also increased the expression of branched-chain amino acid aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKDC) enzyme complexes that regulate the metabolism of BCAAs. Flies with the Bcat heterozygous mutation were not vulnerable to starvation stress, even when exposed to dim LAN, and hemolymph BCAA levels did not decrease in these flies. Furthermore, the vulnerability to starvation stress was also suppressed when the Bcat expression level was reduced in the whole body, neurons, or fat body during adulthood using conditional GAL4 and RNA interference. Finally, the metabolic vulnerability was reversed when BCAAs were fed to wild-type flies exposed to LAN. Thus, short-term dim light exposure at night affects the expression of circadian genes and BCAA metabolism in Drosophila, implying a novel function of BCAAs in suppressing metabolic stress caused by disrupted circadian rhythm.

Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination.

VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of our findings in PD, we identify a missense mutation of Parkin discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.

CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway.

The Hippo pathway controls organ size and tissue homeostasis through the regulation of cell proliferation and apoptosis. However, the exact molecular mechanisms underpinning Hippo pathway regulation are not fully understood. Here, we identify a new component of the Hippo pathway: coronin 7 (CORO7), a coronin protein family member that is involved in organization of the actin cytoskeleton. pod1, the Drosophila ortholog of CORO7, genetically interacts with key Hippo pathway genes in Drosophila. In mammalian cells, CORO7 is required for the activation of the Hippo pathway in response to cell-cell contact, serum deprivation, and cytoskeleton damage. CORO7 forms a complex with the core components of the pathway and functions as a scaffold for the Hippo core kinase complex. Collectively, these results demonstrate that CORO7 is a key scaffold controlling the Hippo pathway via modulating protein-protein interactions.

Drosophila ADCK1 is critical for maintaining mitochondrial structures and functions in the muscle.

The function of AarF domain-containing kinase 1 (ADCK1) has not been thoroughly revealed. Here we identified that ADCK1 utilizes YME1-like 1 ATPase (YME1L1) to control optic atrophy 1 (OPA1) and inner membrane mitochondrial protein (IMMT) in regulating mitochondrial dynamics and cristae structure. We firstly observed that a serious developmental impairment occurred in Drosophila ADCK1 (dADCK1) deletion mutant, resulting in premature death before adulthood. By using temperature sensitive ubiquitously expression driver tub-Gal80ts/tub-Gal4 or muscle-specific expression driver mhc-Gal4, we observed severely defective locomotive activities and structural abnormality in the muscle along with increased mitochondrial fusion in the dADCK1 knockdown flies. Moreover, decreased mitochondrial membrane potential, ATP production and survival rate along with increased ROS and apoptosis in the flies further demonstrated that the structural abnormalities of mitochondria induced by dADCK1 knockdown led to their functional abnormalities. Consistent with the ADCK1 loss-of-function data in Drosophila, ADCK1 over-expression induced mitochondrial fission and clustering in addition to destruction of the cristae structure in Drosophila and mammalian cells. Interestingly, knockdown of YME1L1 rescued the phenotypes of ADCK1 over-expression. Furthermore, genetic epistasis from fly genetics and mammalian cell biology experiments led us to discover the interactions among IMMT, OPA1 and ADCK1. Collectively, these results established a mitochondrial signaling pathway composed of ADCK1, YME1L1, OPA1 and IMMT, which has essential roles in maintaining mitochondrial morphologies and functions in the muscle.

An Inverse Agonist GSK5182 Increases Protein Stability of the Orphan Nuclear Receptor ERRγ via Inhibition of Ubiquitination.

The orphan nuclear receptor, estrogen-related receptor γ (ERRγ) is a constitutively active transcription factor involved in mitochondrial metabolism and energy homeostasis. GSK5182, a specific inverse agonist of ERRγ that inhibits transcriptional activity, induces a conformational change in ERRγ, resulting in a loss of coactivator binding. However, the molecular mechanism underlying the stabilization of the ERRγ protein by its inverse agonist remains largely unknown. In this study, we found that GSK5182 inhibited ubiquitination of ERRγ, thereby stabilizing the ERRγ protein, using cell-based assays and confocal image analysis. Y326 of ERRγ was essential for stabilization by GSK5182, as ligand-induced stabilization of ERRγ was not observed with the ERRγ-Y326A mutant. GSK5182 suppressed ubiquitination of ERRγ by the E3 ligase Parkin and subsequent degradation. The inhibitory activity of GSK5182 was strong even when the ERRγ protein level was elevated, as ERRγ bound to GSK5182 recruited a corepressor, small heterodimer partner-interacting leucine zipper (SMILE), through the activation function 2 (AF-2) domain, without alteration of the nuclear localization or DNA-binding ability of ERRγ. In addition, the AF-2 domain of ERRγ was critical for the regulation of protein stability. Mutants in the AF-2 domain were present at higher levels than the wild type in the absence of GSK5182. Furthermore, the ERRγ-L449A/L451A mutant was no longer susceptible to GSK5182. Thus, the AF-2 domain of ERRγ is responsible for the regulation of transcriptional activity and protein stability by GSK5182. These findings suggest that GSK5182 regulates ERRγ by a unique molecular mechanism, increasing the inactive form of ERRγ via inhibition of ubiquitination.

Von Hippel-Lindau tumor suppressor (VHL) stimulates TOR signaling by interacting with phosphoinositide 3-kinase (PI3K).

Cell growth is positively controlled by the phosphoinositide 3-kinase (PI3K)-target of rapamycin (TOR) signaling pathway under conditions of abundant growth factors and nutrients. To discover additional mechanisms that regulate cell growth, here we performed RNAi-based mosaic analyses in the Drosophila fat body, the primary metabolic organ in the fly. Unexpectedly, the knockdown of the Drosophila von Hippel-Lindau (VHL) gene markedly decreased cell size and body size. These cell growth phenotypes induced by VHL loss of function were recovered by activation of TOR signaling in Drosophila Consistent with the genetic interactions between VHL and the signaling components of PI3K-TOR pathway in Drosophila, we observed that VHL loss of function in mammalian cells causes decreased phosphorylation of ribosomal protein S6 kinase and Akt, which represent the main activities of this pathway. We further demonstrate that VHL activates TOR signaling by directly interacting with the p110 catalytic subunit of PI3K. On the basis of the evolutionarily conserved regulation of PI3K-TOR signaling by VHL observed here, we propose that VHL plays an important role in the regulation and maintenance of proper cell growth in metazoans.

Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.

Mitophagy has been implicated in mitochondrial quality control and in various human diseases. However, the study of in vivo mitophagy remains limited. We previously explored in vivo mitophagy using a transgenic mouse expressing the mitochondria-targeted fluorescent protein Keima (mt-Keima). Here, we generated mt-Keima Drosophila to extend our efforts to study mitophagy in vivo. A series of experiments confirmed that mitophagy can be faithfully and quantitatively measured in mt-Keima Drosophila. We also showed that alterations in mitophagy upon environmental and genetic perturbation can be measured in mt-Keima Drosophila. Analysis of different tissues revealed a variation in basal mitophagy levels in Drosophila tissues. In addition, we found a significant increase in mitophagy levels during Drosophila embryogenesis. Importantly, loss-of-function genetic analysis demonstrated that the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)-Parkin pathway is essential for the induction of mitophagy in vivo in response to hypoxic exposure and rotenone treatment. These studies showed that the mt-Keima Drosophila system is a useful tool for understanding the role and molecular mechanism of mitophagy in vivo. In addition, we demonstrated the essential role of the PINK1-Parkin pathway in mitophagy induction in response to mitochondrial dysfunction.-Kim, Y. Y., Um, J.-H., Yoon, J.-H., Kim, H., Lee, D.-Y., Lee, Y. J., Jee, H. J., Kim, Y. M., Jang, J. S., Jang, Y.-G., Chung, J., Park, H. T., Finkel, T., Koh, H., Yun, J. Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.

Isocitrate protects DJ-1 null dopaminergic cells from oxidative stress through NADP+-dependent isocitrate dehydrogenase (IDH).

DJ-1 is one of the causative genes for early onset familiar Parkinson's disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP+-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of IDH induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in Drosophila, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using Drosophila and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian DJ-1 null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with DJ-1 dysfunction.

Cereblon Maintains Synaptic and Cognitive Function by Regulating BK Channel.

Mutations in the cereblon (CRBN) gene cause human intellectual disability, one of the most common cognitive disorders. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. We investigated the role of CRBN in synaptic function and animal behavior using male mouse and Drosophila models. Crbn knock-out (KO) mice showed normal brain and spine morphology as well as intact synaptic plasticity; however, they also exhibited decreases in synaptic transmission and presynaptic release probability exclusively in excitatory synapses. Presynaptic function was impaired not only by loss of CRBN expression, but also by expression of pathogenic CRBN mutants (human R419X mutant and Drosophila G552X mutant). We found that the BK channel blockers paxilline and iberiotoxin reversed this decrease in presynaptic release probability in Crbn KO mice. In addition, paxilline treatment also restored normal cognitive behavior in Crbn KO mice. These results strongly suggest that increased BK channel activity is the pathological mechanism of intellectual disability in CRBN mutations.SIGNIFICANCE STATEMENTCereblon (CRBN), a well known target of the immunomodulatory drug thalidomide, was originally identified as a gene that causes human intellectual disability when mutated. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. Based on the idea that synaptic abnormalities are the most common factor in cognitive dysfunction, we monitored the synaptic structure and function of Crbn knock-out (KO) animals to identify the molecular mechanisms of intellectual disability. Here, we found that Crbn KO animals showed cognitive deficits caused by enhanced BK channel activity and reduced presynaptic glutamate release. Our findings suggest a physiological pathomechanism of the intellectual disability-related gene CRBN and will contribute to the development of therapeutic strategies for CRBN-related intellectual disability.

ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled.

Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/ß-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with ß-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/ß-catenin signaling pathway by phosphorylating Dsh.

DNA damage-induced RORα is crucial for p53 stabilization and increased apoptosis.

A critical component of the DNA damage response is the p53 tumor suppressor, and aberrant p53 function leads to uncontrolled cell proliferation and malignancy. Several molecules have been shown to regulate p53 stability; however, genome-wide systemic approaches for determining the affected, specific downstream target genes have not been extensively studied. Here, we first identified an orphan nuclear receptor, RORα, as a direct target gene of p53, which contains functional p53 response elements. The functional consequences of DNA damage-induced RORα are to stabilize p53 and activate p53 transcription in a HAUSP/Usp7-dependent manner. Interestingly, microarray analysis revealed that RORα-mediated p53 stabilization leads to the activation of a subset of p53 target genes that are specifically involved in apoptosis. We further confirmed that RORα enhances p53-dependent, in vivo apoptotic function in the Drosophila model system. Together, we determined that RORα is a p53 regulator that exerts its role in increased apoptosis via p53.

Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death.

Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.

Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila.

LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. Here we show that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, we identify HDAC4, a class IIa histone deacetylase, as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses. We also identify that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila.

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity.

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.

Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) Mutation and TRAP1 Inhibitor Gamitrinib-triphenylphosphonium (G-TPP) Induce a Forkhead Box O (FOXO)-dependent Cell Protective Signal from Mitochondria.

TRAP1 (tumor necrosis factor receptor-associated protein 1), a mitochondrial Hsp90 family chaperone, has been identified as a critical regulator of cell survival and bioenergetics in tumor cells. To discover novel signaling networks regulated by TRAP1, we generated Drosophila TRAP1 mutants. The mutants successfully developed into adults and produced fertile progeny, showing that TRAP1 is dispensable in development and reproduction. Surprisingly, mutation or knockdown of TRAP1 markedly enhanced Drosophila survival under oxidative stress. Moreover, TRAP1 mutation ameliorated mitochondrial dysfunction and dopaminergic (DA) neuron loss induced by deletion of a familial Parkinson disease gene PINK1 (Pten-induced kinase 1) in Drosophila. Gamitrinib-triphenylphosphonium, a mitochondria-targeted Hsp90 inhibitor that increases cell death in HeLa and MCF7 cells, consistently inhibited cell death induced by oxidative stress and mitochondrial dysfunction induced by PINK1 mutation in mouse embryonic fibroblast cells and DA cell models such as SH-SY5Y and SN4741 cells. Additionally, gamitrinib-triphenylphosphonium also suppressed the defective locomotive activity and DA neuron loss in Drosophila PINK1 null mutants. In further genetic analyses, we showed enhanced expression of Thor, a downstream target gene of transcription factor FOXO, in TRAP1 mutants. Furthermore, deletion of FOXO almost nullified the protective roles of TRAP1 mutation against oxidative stress and PINK1 mutation. These results strongly suggest that inhibition of the mitochondrial chaperone TRAP1 generates a retrograde cell protective signal from mitochondria to the nucleus in a FOXO-dependent manner.

Drosophila Low Temperature Viability Protein 1 (LTV1) Is Required for Ribosome Biogenesis and Cell Growth Downstream of Drosophila Myc (dMyc).

During animal development, various signaling pathways converge to regulate cell growth. In this study, we identified LTV1 as a novel cell growth regulator in Drosophila. LTV1 mutant larvae exhibited developmental delays and lethality at the second larval stage. Using biochemical studies, we discovered that LTV1 interacted with ribosomal protein S3 and co-purified with free 40S ribosome subunits. We further demonstrated that LTV1 is crucial for ribosome biogenesis through 40S ribosome subunit synthesis and preribosomal RNA processing, suggesting that LTV1 is required for cell growth by regulating protein synthesis. We also demonstrated that Drosophila Myc (dMyc) directly regulates LTV1 transcription and requires LTV1 to stimulate ribosome biogenesis. Importantly, the loss of LTV1 blocked the cell growth and endoreplication induced by dMyc. Combined, these results suggest that LTV1 is a key downstream factor of dMyc-induced cell growth by properly maintaining ribosome biogenesis.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

DJ-1, a Parkinson's disease (PD)-associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1ß, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1ß mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1ß mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1ß mutants and that dFOXO activity was increased in the heads of DJ-1ß mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1ß mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1ß under oxidative stress conditions; and, furthermore, targeted expression of DJ-1ß to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1ß protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

Crif1 deficiency reduces adipose OXPHOS capacity and triggers inflammation and insulin resistance in mice.

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS-deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA-encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.