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Sample records for 60s biogenesis pathway

  1. The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology

    PubMed Central

    Wandrey, Franziska; Montellese, Christian; Koos, Krisztian; Badertscher, Lukas; Bammert, Lukas; Cook, Atlanta G.; Zemp, Ivo; Horvath, Peter

    2015-01-01

    The interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis. We show that association of the NF45/NF90 heterodimer with pre-60S ribosomal particles requires the double-stranded RNA binding domains of NF90, while depletion of NF45 and NF90 by RNA interference leads to a defect in 60S biogenesis. Nucleoli of cells depleted of NF45 and NF90 have altered morphology and display a characteristic spherical shape. These effects are not due to impaired rRNA transcription or processing of the precursors to 28S rRNA. Consistent with a role of the NF45/NF90 heterodimer in nucleolar steps of 60S subunit biogenesis, downregulation of NF45 and NF90 leads to a p53 response, accompanied by induction of the cyclin-dependent kinase inhibitor p21/CIP1, which can be counteracted by depletion of RPL11. Together, these data indicate that NF45 and NF90 are novel higher-eukaryote-specific factors required for the maturation of 60S ribosomal subunits. PMID:26240280

  2. Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation.

    PubMed

    Greber, Basil Johannes; Gerhardy, Stefan; Leitner, Alexander; Leibundgut, Marc; Salem, Michèle; Boehringer, Daniel; Leulliot, Nicolas; Aebersold, Ruedi; Panse, Vikram Govind; Ban, Nenad

    2016-01-14

    Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation. PMID:26709046

  3. Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits.

    PubMed Central

    Adams, Cynthia C; Jakovljevic, Jelena; Roman, Judibelle; Harnpicharnchai, Piyanun; Woolford, John L

    2002-01-01

    To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A. PMID:11911362

  4. MicroRNA biogenesis pathways in cancer

    PubMed Central

    Lin, Shuibin; Gregory, Richard I.

    2016-01-01

    MicroRNAs (miRNAs) are critical regulators of gene expression. Amplification and overexpression of individual ‘oncomiRs’ or genetic loss of tumour suppressor miRNAs are associated with human cancer and are sufficient to drive tumorigenesis in mouse models. Furthermore, global miRNA depletion caused by genetic and epigenetic alterations in components of the miRNA biogenesis machinery is oncogenic. This, together with the recent identification of novel miRNA regulatory factors and pathways, highlights the importance of miRNA dysregulation in cancer. PMID:25998712

  5. SIGNALING PATHWAYS IN MELANOSOME BIOGENESIS AND PATHOLOGY

    PubMed Central

    Schiaffino, Maria Vittoria

    2010-01-01

    Melanosomes are the specialized intracellular organelles of pigment cells devoted to the synthesis, storage and transport of melanin pigments, which are responsible for most visible pigmentation in mammals and other vertebrates. As a direct consequence, any genetic mutation resulting in alteration of melanosomal function, either because affecting pigment cell survival, migration and differentiation, or because interfering with melanosome biogenesis, transport and transfer to keratinocytes, is immediately translated into color variations of skin, fur, hair or eyes. Thus, over one hundred genes and proteins have been identified as pigmentary determinants in mammals, providing us with a deep understanding of this biological system, which functions by using mechanisms and processes that have parallels in other tissues and organs. In particular, many genes implicated in melanosome biogenesis have been characterized, so that melanosomes represent an incredible source of information and a model for organelles belonging to the secretory pathway. Furthermore, the function of melanosomes can be associated with common physiological phenotypes, such as variation of pigmentation among individuals, and with rare pathological conditions, such as albinism, characterized by severe visual defects. Among the most relevant mechanisms operating in melanosome biogenesis are the signal transduction pathways mediated by two peculiar G protein-coupled receptors: the melanocortin-1 receptor (MC1R), involved in the fair skin/red hair phenotype and skin cancer; and OA1 (GPR143), whose loss-of-function results in X-linked ocular albinism. This review will focus on the most recent novelties regarding the functioning of these two receptors, by highlighting emerging signaling mechanisms and general implications for cell biology and pathology. PMID:20381640

  6. Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis.

    PubMed

    Sulima, Sergey O; Patchett, Stephanie; Advani, Vivek M; De Keersmaecker, Kim; Johnson, Arlen W; Dinman, Jonathan D

    2014-04-15

    Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia. This mutation causes a late-stage 60S subunit maturation failure that targets mutant ribosomes for degradation. The resulting deficit in ribosomes causes the hypoproliferative phenotype. This 60S subunit shortage, in turn, exerts pressure on cells to select for suppressors of the ribosome biogenesis defect, allowing them to reestablish normal levels of ribosome production and cell proliferation. However, suppression at this step releases structurally and functionally defective ribosomes into the translationally active pool, and the translational fidelity defects of these mutants culminate in destabilization of selected mRNAs and shortened telomeres. We suggest that in exchange for resolving their short-term ribosome deficits through compensatory trans-acting suppressors, cells are penalized in the long term by changes in gene expression that ultimately undermine cellular homeostasis. PMID:24706786

  7. Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway.

    PubMed

    Thomas, Franziska; Kutay, Ulrike

    2003-06-15

    The production of ribosomes constitutes a major biosynthetic task for cells. Eukaryotic small and large ribosomal subunits are assembled in the nucleolus and independently exported to the cytoplasm. Most nuclear export pathways require RanGTP-binding export receptors. We analyzed the role of CRM1, the export receptor for leucine-rich nuclear export signals (NES), in the biogenesis of ribosomal subunits in vertebrate cells. Inhibition of the CRM1 export pathway led to a defect in nuclear export of both 40S and 60S subunits in HeLa cells. Moreover, the export of newly made ribosomal subunits in Xenopus oocytes was efficiently and specifically competed by BSA-NES conjugates. The CRM1 dependence of 60S subunit export suggested a conserved function for NMD3, a factor proposed to be a 60S subunit export adaptor in yeast. Indeed, we observed that nuclear export of human NMD3 (hNMD3) is sensitive to leptomycin B (LMB), which inactivates CRM1. It had, however, not yet been demonstrated that Nmd3 can interact with CRM1. Using purified recombinant proteins we have shown here that hNMD3 binds to CRM1 directly, in a RanGTP-dependent manner, by way of a C-terminal NES sequence. Our results suggest that the functions of CRM1 and NMD3 in ribosomal subunit export are conserved from yeast to higher eukaryotes. PMID:12724356

  8. Biogenesis of γ-secretase early in the secretory pathway

    PubMed Central

    Kim, Jinoh; Kleizen, Bertrand; Choy, Regina; Thinakaran, Gopal; Sisodia, Sangram S.; Schekman, Randy W.

    2007-01-01

    γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology. PMID:18056412

  9. Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

    PubMed Central

    Li, Zhihua; Lee, Insuk; Moradi, Emily; Hung, Nai-Jung; Johnson, Arlen W.; Marcotte, Edward M.

    2009-01-01

    Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process. PMID:19806183

  10. Syringaresinol induces mitochondrial biogenesis through activation of PPARβ pathway in skeletal muscle cells.

    PubMed

    Thach, Trung Thanh; Lee, Chan-Kyu; Park, Hyun Woo; Lee, Sang-Jun; Lee, Sung-Joon

    2016-08-15

    Activation of peroxisome proliferator-activated receptors (PPARs) plays a crucial role in cellular energy metabolism that directly impacts mitochondrial biogenesis. In this study, we demonstrate that syringaresinol, a pharmacological lignan extracted from Panax ginseng berry, moderately binds to and activates PPARβ with KD and EC50 values of 27.62±15.76μM and 18.11±4.77μM, respectively. Subsequently, the expression of peroxisome proliferator-activated receptor γ coactivator-1α together with PPARβ transcriptional targets, mitochondrial carnitine palmitoyltransferase 1 and uncoupling protein 2, was also enhanced in terms of both mRNA and protein levels. The activation of these proteins induced mitochondrial biogenesis by enrichment of mitochondrial replication and density within C2C12 myotubes. Importantly, knockdown of PPARβ reduced the syringaresinol-induced protein expression followed by the significant reduction of mitochondrial biogenesis. Taken together, our results indicate that syringaresinol induces mitochondrial biogenesis by activating PPARβ pathway. PMID:27450788

  11. Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway.

    PubMed

    Boyd, Eric S; Thomas, Khaleh M; Dai, Yuyuan; Boyd, Jeff M; Outten, F Wayne

    2014-09-23

    Iron-sulfur (Fe-S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe-S clusters and the fundamental requirement for Fe-S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth's atmosphere. Intriguingly, Fe-S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe-S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe-S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe-S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB-SufC scaffold complex. This analysis provides a new framework for the study of Fe-S cluster biogenesis pathways and Fe-S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen. PMID:25153801

  12. Interplay between Oxygen and Fe–S Cluster Biogenesis: Insights from the Suf Pathway

    PubMed Central

    2015-01-01

    Iron–sulfur (Fe–S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe–S clusters and the fundamental requirement for Fe–S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth’s atmosphere. Intriguingly, Fe–S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe–S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe–S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe–S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB–SufC scaffold complex. This analysis provides a new framework for the study of Fe–S cluster biogenesis pathways and Fe–S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen. PMID:25153801

  13. Recent advances in the Suf Fe-S cluster biogenesis pathway: Beyond the Proteobacteria

    PubMed Central

    Outten, F. Wayne

    2014-01-01

    Fe-S clusters play critical roles in cellular function throughout all three kingdoms of life. Consequently, Fe-S cluster biogenesis systems are present in most organisms. The Suf (sulfur formation) system is the most ancient of the three characterized Fe-S cluster biogenesis pathways, which also include the Isc and Nif systems. Much of the first work on the Suf system took place in Gram-negative Proteobacteria used as model organisms. These early studies led to a wealth of biochemical, genetic, and physiological information on Suf function. From those studies we have learned that SufB functions as an Fe-S scaffold in conjunction with SufC (and in some cases SufD). SufS and SufE together mobilize sulfur for cluster assembly and SufA traffics the complete Fe-S cluster from SufB to target apo-proteins. However, recent progress on the Suf system in other organisms has opened up new avenues of research and new hypotheses about Suf function. This review focuses primarily on the most recent discoveries about the Suf pathway and where those new models may lead the field. PMID:25447545

  14. Diversity of the piRNA pathway for nonself silencing: worm-specific piRNA biogenesis factors

    PubMed Central

    Izumi, Natsuko; Tomari, Yukihide

    2014-01-01

    The PIWI-interacting RNA (piRNA) pathway protects animal germline cells from transposable elements and other genomic invaders. Although the genome defense function of piRNAs has been well established, the mechanisms of their biogenesis remain poorly understood. In this issue of Genes & Development, three groups identify novel factors required for piRNA biogenesis in Caenorhabditis elegans. These works greatly expand our understanding of the piRNA pathway in worms, highlighting both its shared and its unique properties. PMID:24696451

  15. Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit.

    PubMed

    Mulder, Anke M; Yoshioka, Craig; Beck, Andrea H; Bunner, Anne E; Milligan, Ronald A; Potter, Clinton S; Carragher, Bridget; Williamson, James R

    2010-10-29

    Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the Escherichia coli 30S subunit suggest that ribosome assembly occurs via multiple parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery single-particle profiling (DSP), an application of time-resolved electron microscopy, was used to obtain more than 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome-assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates. PMID:21030658

  16. A novel pathway for outer membrane protein biogenesis in Gram‐negative bacteria

    PubMed Central

    Jeeves, Mark

    2015-01-01

    Summary The understanding of the biogenesis of the outer membrane of Gram‐negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram‐negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram‐negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β‐barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram‐negative outer membrane that is independent of the BAM complex and TAM. PMID:26059329

  17. A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria.

    PubMed

    Jeeves, Mark; Knowles, Timothy J

    2015-08-01

    The understanding of the biogenesis of the outer membrane of Gram-negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram-negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β-barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram-negative outer membrane that is independent of the BAM complex and TAM. PMID:26059329

  18. Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

    PubMed Central

    Titorenko, V I; Ogrydziak, D M; Rachubinski, R A

    1997-01-01

    We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum. PMID:9271399

  19. A role of uridylation pathway for blockade of let-7 microRNA biogenesis by Lin28B

    PubMed Central

    Suzuki, Hiroshi I; Katsura, Akihiro; Miyazono, Kohei

    2015-01-01

    The precise control of microRNA (miRNA) biosynthesis is crucial for gene regulation. Lin28A and Lin28B are selective inhibitors of biogenesis of let-7 miRNAs involved in development and tumorigenesis. Lin28A selectively inhibits let-7 biogenesis through cytoplasmic uridylation of precursor let-7 by TUT4 terminal uridyl transferase and subsequent degradation by Dis3l2 exonuclease. However, a role of this uridylation pathway remains unclear in let-7 blockade by Lin28B, a paralog of Lin28A, while Lin28B is reported to engage a distinct mechanism in the nucleus to suppress let-7. Here we revisit a functional link between Lin28B and the uridylation pathway with a focus on let-7 metabolism in cancer cells. Both Lin28A and Lin28B interacted with Dis3l2 in the cytoplasm, and silencing of Dis3l2 upregulated uridylated pre-let-7 in both Lin28A- and Lin28B-expressing cancer cell lines. In addition, we found that amounts of let-7 precursors influenced intracellular localization of Lin28B. Furthermore, we found that MCPIP1 (Zc3h12a) ribonuclease was also involved in degradation of both non-uridylated and uridylated pre-let-7. Cancer transcriptome analysis showed association of expression levels of Lin28B and uridylation pathway components, TUT4 and Dis3l2, in various human cancer cells and hepatocellular carcinoma. Collectively, these results suggest that cytoplasmic uridylation pathway actively participates in blockade of let-7 biogenesis by Lin28B. PMID:26080928

  20. The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.

    PubMed

    Cwiklinski, Krystyna; de la Torre-Escudero, Eduardo; Trelis, Maria; Bernal, Dolores; Dufresne, Philippe J; Brennan, Gerard P; O'Neill, Sandra; Tort, Jose; Paterson, Steve; Marcilla, Antonio; Dalton, John P; Robinson, Mark W

    2015-12-01

    Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. PMID:26486420

  1. Carvedilol promotes mitochondrial biogenesis by regulating the PGC-1/TFAM pathway in human umbilical vein endothelial cells (HUVECs).

    PubMed

    Yao, Kai; Zhang, Wayne W; Yao, Luyu; Yang, Shu; Nie, Wanpin; Huang, Feizhou

    2016-02-19

    Carvedilol, a third-generation and nonselective β-adrenoceptor antagonist, is a licensed drug for treating patients suffering from heart failure in clinics. It has been shown that Carvedilol protects cells against mitochondrial dysfunction. However, it's unknown whether Carvedilol affects mitochondrial biogenesis. In this study, we found that treatment with Carvedilol in HUVECs resulted in a significant increase of PGC-1α, NRF1, and TFAM. Notably, Carvedilol significantly increased mtDNA contents and the two mitochondrial proteins, cytochrome C and COX IV. In addition, MitoTracker Red staining results indicated that treatment with Carvedilol increased mitochondria mass. Mechanistically, we found that the effect of Carvedilol on the expression of PGC-1α is mediated by the PKA-CREB pathway. Importantly, our results revealed that stimulation of mitochondrial biogenesis by carvedilol resulted in functional gain of the mitochondria by showing increased oxygen consumption and mitochondrial respiratory rate. The increased expression of PGC-1α and mitochondrial biogenesis induced by Carvedilol might suggest a new mechanism of the therapeutic effects of Carvedilol in heart failure. PMID:26797282

  2. The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis*

    PubMed Central

    Cwiklinski, Krystyna; de la Torre-Escudero, Eduardo; Trelis, Maria; Bernal, Dolores; Dufresne, Philippe J.; Brennan, Gerard P.; O'Neill, Sandra; Tort, Jose; Paterson, Steve; Marcilla, Antonio; Dalton, John P.; Robinson, Mark W.

    2015-01-01

    Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. PMID:26486420

  3. Modulators of the microRNA biogenesis pathway via arrayed lentiviral enabled RNAi screening for drug and biomarker discovery

    PubMed Central

    Shum, David; Bhinder, Bhavneet; Djaballah, Hakim

    2013-01-01

    MicroRNAs (miRNAs) are small endogenous and conserved non-coding RNA molecules that regulate gene expression. Although the first miRNA was discovered well over sixteen years ago, little is known about their biogenesis and it is only recently that we have begun to understand their scope and diversity. For this purpose, we performed an RNAi screen aimed at identifying genes involved in their biogenesis pathway with a potential use as biomarkers. Using a previously developed miRNA 21 (miR-21) EGFP-based biosensor cell based assay monitoring green fluorescence enhancements, we performed an arrayed short hairpin RNA (shRNA) screen against a lentiviral particle ready TRC1 library covering 16,039 genes in 384-well plate format, and interrogating the genome one gene at a time building a panoramic view of endogenous miRNA activity. Using the BDA method for RNAi data analysis, we nominate 497 gene candidates the knockdown of which increased the EGFP fluorescence and yielding an initial hit rate of 3.09%; of which only 22, with reported validated clones, are deemed high-confidence gene candidates. An unexpected and surprising result was that only DROSHA was identified as a hit out of the seven core essential miRNA biogenesis genes; suggesting that perhaps intracellular shRNA processing into the correct duplex may be cell dependent and with differential outcome. Biological classification revealed several major control junctions among them genes involved in transport and vesicular trafficking. In summary, we report on 22 high confidence gene candidate regulators of miRNA biogenesis with potential use in drug and biomarker discovery. PMID:23977983

  4. Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway.

    PubMed

    Kurz, M; Martin, H; Rassow, J; Pfanner, N; Ryan, M T

    1999-07-01

    Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP. PMID:10397776

  5. Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

    PubMed Central

    Joffe, Luna S.; Guimarães, Allan J.; Sobreira, Tiago J. P.; Nosanchuk, Joshua D.; Cordero, Radames J. B.; Frases, Susana; Casadevall, Arturo; Almeida, Igor C.; Nimrichter, Leonardo; Rodrigues, Marcio L.

    2010-01-01

    Background Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. Conclusions/Significance Our results suggest that both conventional and unconventional pathways of secretion are required for

  6. MicroRNA biogenesis pathway from the salmon louse (Caligus rogercresseyi): emerging role in delousing drug response.

    PubMed

    Valenzuela-Miranda, Diego; Nuñez-Acuña, Gustavo; Valenzuela-Muñoz, Valentina; Asgari, Sassan; Gallardo-Escárate, Cristian

    2015-01-25

    Despite the increasing evidence of the importance of microRNAs (miRNAs) in the regulation of multiple biological processes, the molecular bases supporting this regulation are still barely understood in crustaceans. Therefore, the molecular characterization and transcriptome modulation of the miRNA biogenesis pathway were evaluated in the salmon louse Caligus rogercresseyi, an ectoparasite that constitutes one of the biggest concerns for salmonid aquaculture industry. Hence, RNA-Seq analysis was conducted from six different developmental stages, and also after bioassays with delousing drugs Deltamethrin and Azamethiphos using adult individuals. In silico analysis evidenced 24 putative genes involved in the miRNA pathway such as biogenesis, transport, maturation and miRNA-target interaction. Moreover, 243 putative single nucleotide polymorphisms (SNPs) were identified, 15 of which showed non-synonym mutations. RNA-Seq analysis revealed that CCR4-Not complex subunit 3 (CNOT3) was upregulated at earlier developmental stages (nauplius I-II and copepodid), and also after the exposure to Azamethiphos, but not to Deltamethrin. In contrast, the subunit 7 (CNOT7) showed an inverse expression pattern. Different Argonaute transcripts were associated to chalimus and adult stages, revealing specific expression patterns in response to antiparasitic drugs. Our results suggest novel insights into the regulatory network of the post-transcriptional gene regulation in C. rogercresseyi mediated by miRNAs, evidencing a putative role during the ontogeny and drug response. PMID:25447902

  7. Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili

    PubMed Central

    Wolfgang, Matthew; van Putten, Jos P.M.; Hayes, Stanley F.; Dorward, David; Koomey, Michael

    2000-01-01

    Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components. PMID:11101514

  8. An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway

    PubMed Central

    Shum, David; Bhinder, Bhavneet; Ramirez, Christina N.; Radu, Constantin; Calder, Paul A.; Beauchamp, Lesslie; Farazi, T.; Landthaler, M.; Tuschi, T.; Magdaleno, Susan

    2013-01-01

    Abstract MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder–Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function. PMID:23153064

  9. GAIP Interacting Protein C-Terminus Regulates Autophagy and Exosome Biogenesis of Pancreatic Cancer through Metabolic Pathways

    PubMed Central

    Bhattacharya, Santanu; Pal, Krishnendu; Sharma, Anil K.; Dutta, Shamit K.; Lau, Julie S.; Yan, Irene K.; Wang, Enfeng; Elkhanany, Ahmed; Alkharfy, Khalid M.; Sanyal, Arunik; Patel, Tushar C.; Chari, Suresh T.; Spaller, Mark R.; Mukhopadhyay, Debabrata

    2014-01-01

    GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer. PMID:25469510

  10. Evidence of a Ca(2+)-(*)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vieira, André L G; Linares, Edlaine; Augusto, Ohara; Gomes, Suely L

    2009-08-01

    The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(*)NO-cGMP signaling pathway playing a role in zoospore biogenesis. PMID:19393757

  11. A Dicer-independent miRNA biogenesis pathway that requires Ago catalysis

    PubMed Central

    Cheloufi, Sihem; Dos Santos, Camila O.; Chong, Mark M. W.; Hannon, Gregory J.

    2010-01-01

    The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3′ end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development. PMID:20424607

  12. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis.

    PubMed

    Cheloufi, Sihem; Dos Santos, Camila O; Chong, Mark M W; Hannon, Gregory J

    2010-06-01

    The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3' end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development. PMID:20424607

  13. The CDP-Ethanolamine Pathway Regulates Skeletal Muscle Diacylglycerol Content and Mitochondrial Biogenesis without Altering Insulin Sensitivity.

    PubMed

    Selathurai, Ahrathy; Kowalski, Greg M; Burch, Micah L; Sepulveda, Patricio; Risis, Steve; Lee-Young, Robert S; Lamon, Severine; Meikle, Peter J; Genders, Amanda J; McGee, Sean L; Watt, Matthew J; Russell, Aaron P; Frank, Matthew; Jackowski, Suzanne; Febbraio, Mark A; Bruce, Clinton R

    2015-05-01

    Accumulation of diacylglycerol (DG) in muscle is thought to cause insulin resistance. DG is a precursor for phospholipids, thus phospholipid synthesis could be involved in regulating muscle DG. Little is known about the interaction between phospholipid and DG in muscle; therefore, we examined whether disrupting muscle phospholipid synthesis, specifically phosphatidylethanolamine (PtdEtn), would influence muscle DG content and insulin sensitivity. Muscle PtdEtn synthesis was disrupted by deleting CTP:phosphoethanolamine cytidylyltransferase (ECT), the rate-limiting enzyme in the CDP-ethanolamine pathway, a major route for PtdEtn production. While PtdEtn was reduced in muscle-specific ECT knockout mice, intramyocellular and membrane-associated DG was markedly increased. Importantly, however, this was not associated with insulin resistance. Unexpectedly, mitochondrial biogenesis and muscle oxidative capacity were increased in muscle-specific ECT knockout mice and were accompanied by enhanced exercise performance. These findings highlight the importance of the CDP-ethanolamine pathway in regulating muscle DG content and challenge the DG-induced insulin resistance hypothesis. PMID:25955207

  14. Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis

    PubMed Central

    Pakharukova, Natalia; Garnett, James A.; Tuittila, Minna; Paavilainen, Sari; Diallo, Mamou; Xu, Yingqi; Matthews, Steve J.; Zavialov, Anton V.

    2015-01-01

    Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems. PMID:26587649

  15. Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis.

    PubMed

    Pakharukova, Natalia; Garnett, James A; Tuittila, Minna; Paavilainen, Sari; Diallo, Mamou; Xu, Yingqi; Matthews, Steve J; Zavialov, Anton V

    2015-11-01

    Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems. PMID:26587649

  16. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

    PubMed Central

    Levin, David E.

    2011-01-01

    The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

  17. The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway

    PubMed Central

    Õunap, Kadri; Leetsi, Lilian; Matsoo, Maarja; Kurg, Reet

    2015-01-01

    The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells. PMID:26214185

  18. Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

    PubMed

    Sinzel, Monika; Tan, Tao; Wendling, Philipp; Kalbacher, Hubert; Özbalci, Cagakan; Chelius, Xenia; Westermann, Benedikt; Brügger, Britta; Rapaport, Doron; Dimmer, Kai Stefan

    2016-07-01

    Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane. PMID:27226123

  19. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway

    PubMed Central

    Vasale, Jessica J.; Gu, Weifeng; Thivierge, Caroline; Batista, Pedro J; Claycomb, Julie M.; Youngman, Elaine M.; Duchaine, Thomas F.; Mello, Craig C.; Conte, Darryl

    2010-01-01

    Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi, and nematodes, cellular RNA-dependent RNA polymerases (RdRPs) use AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in Caenorhabditis elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4 are required for the biogenesis of 26-nt small RNAs with a 5′ guanine (26G-RNAs) and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a nonrandom distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control overexpression resulting from gene expansion. PMID:20133583

  20. Biogenesis of Y RNA-derived small RNAs is independent of the microRNA pathway.

    PubMed

    Nicolas, Francisco Esteban; Hall, Adam E; Csorba, Tibor; Turnbull, Carly; Dalmay, Tamas

    2012-04-24

    Y RNAs are approximately 100 nucleotide long conserved cytoplasmic non-coding RNAs, which produce smaller RNA fragments during apoptosis. Here we show that these smaller RNA molecules are also produced in non-stressed cells and in a range of human cancerous and non-cancerous cell types. Recent reports have speculated that the cleavage products of Y RNAs enter the microRNA pathway. We tested this hypothesis and found that Y5 and Y3 RNA fragments are Dicer independent, they are in different complexes than microRNAs and that they are not co-immunoprecipitated with Ago2. Therefore we conclude that Y RNA fragments do not enter the microRNA pathway. PMID:22575660

  1. A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

    PubMed Central

    Piek, Susannah; Kahler, Charlene M.

    2012-01-01

    The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism. PMID:23267440

  2. Experimental Genetics of Plasmodium berghei NFU in the Apicoplast Iron-Sulfur Cluster Biogenesis Pathway

    PubMed Central

    Haussig, Joana M.; Matuschewski, Kai; Kooij, Taco W. A.

    2013-01-01

    Eukaryotic pathogens of the phylum Apicomplexa contain a non-photosynthetic plastid, termed apicoplast. Within this organelle distinct iron-sulfur [Fe-S] cluster proteins are likely central to biosynthesis pathways, including generation of isoprenoids and lipoic acid. Here, we targeted a nuclear-encoded component of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria parasite Plasmodium berghei. We show that ablation of the gene encoding a nitrogen fixation factor U (NifU)-like domain containing protein (NFUapi) resulted in parasites that were able to complete the entire life cycle indicating redundant or non-essential functions. nfu– parasites displayed reduced merosome formation in vitro, suggesting that apicoplast NFUapi plays an auxiliary role in establishing a blood stage infection. NFUapi fused to a combined fluorescent protein-epitope tag delineates the Plasmodium apicoplast and was tested to revisit inhibition of liver stage development by azithromycin and fosmidomycin. We show that the branched apicoplast signal is entirely abolished by azithromycin treatment, while fosmidomycin had no effect on apicoplast morphology. In conclusion, our experimental genetics analysis supports specialized and/or redundant role(s) for NFUapi in the [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite. PMID:23805304

  3. An image-based biosensor assay strategy to screen for modulators of the microRNA 21 biogenesis pathway.

    PubMed

    Shum, David; Bhinder, Bhavneet; Radu, Constantin; Farazi, Thalia; Landthaler, Markus; Tuschl, Thomas; Calder, Paul; Ramirez, Christina N; Djaballah, Hakim

    2012-08-01

    microRNAs (miRNAs) are evolutionary conserved, small endogenous non-coding, RNA molecules. Although their mode of action has been extensively studied, little is known about their biogenesis. As their altered expression has been implicated in many diseases, small molecules that would modulate their expression are sought after. They are generated through the concerted action of several complexes which promote their transcription, maturation, export, trafficking, and loading of mature miRNA into silencing complexes. An increasing number of studies have suggested that each of these steps serves as a regulatory junction in the process, and therefore provides an intervention point. For this purpose, we have developed a simple image-based assay strategy to screen for such modulators. Here, we describe its successful implementation which combines the use of a microRNA 21 (miR-21) synthetic mimic together with an EGFP based reporter cell line, where its expression is under the control of miR-21, to monitor EGFP expression in a format suitable for HTS. The strategy was further validated using a small panel of known gene modulators of the miRNA pathway. A screen was performed in duplicate against a library of 6,912 compounds and identified 48 initial positives exhibiting enhanced EGFP fluorescence intensity. 42 compounds were found to be inherently fluorescent in the green channel leaving the remaining 6 as potential inhibitors and with a positive rate of 0.09%. Taken together, this validated strategy offers the opportunity to discover novel and specific inhibitors of the pathway through the screening of diverse chemical libraries. PMID:22540737

  4. Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

    PubMed Central

    Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J.; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F.; Emili, Andrew

    2011-01-01

    As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. PMID:22125496

  5. Cutis laxa: intersection of elastic fiber biogenesis, TGFβ signaling, the secretory pathway and metabolism.

    PubMed

    Urban, Zsolt; Davis, Elaine C

    2014-01-01

    Cutis laxa (CL), a disease characterized by redundant and inelastic skin, displays extensive locus heterogeneity. Together with geroderma osteodysplasticum and arterial tortuosity syndrome, which show phenotypic overlap with CL, eleven CL-related genes have been identified to date, which encode proteins within 3 groups. Elastin, fibulin-4, fibulin-5 and latent transforming growth factor-β-binding protein 4 are secreted proteins which form elastic fibers and are involved in the sequestration and subsequent activation of transforming growth factor-β (TGFβ). Proteins within the second group, localized to the secretory pathway, perform transport and membrane trafficking functions necessary for the modification and secretion of elastic fiber components. Key proteins include a subunit of the vacuolar-type proton pump, which ensures the efficient secretion of tropoelastin, the precursor or elastin. A copper transporter is required for the activity of lysyl oxidases, which crosslink collagen and elastin. A Rab6-interacting goglin recruits kinesin motors to Golgi-vesicles facilitating the transport from the Golgi to the plasma membrane. The Rab and Ras interactor 2 regulates the activity of Rab5, a small guanosine triphosphatase essential for the endocytosis of various cell surface receptors, including integrins. Proteins of the third group related to CL perform metabolic functions within the mitochondria, inhibiting the accumulation of reactive oxygen species. Two of these proteins catalyze subsequent steps in the conversion of glutamate to proline. The third transports dehydroascorbate into mitochondria. Recent studies on CL-related proteins highlight the intricate connections among membrane trafficking, metabolism, extracellular matrix assembly, and TGFβ signaling. PMID:23954411

  6. The ribosome biogenesis pathway as an early target of benzyl butyl phthalate (BBP) toxicity in Chironomus riparius larvae.

    PubMed

    Herrero, Óscar; Planelló, Rosario; Morcillo, Gloria

    2016-02-01

    Butyl benzyl phthalate (BBP) is a ubiquitous contaminant whose presence in the environment is expected for decades, since it has been extensively used worldwide as a plasticizer in the polyvinyl chloride (PVC) industry and the manufacturing of many other products. In the present study, the interaction of BBP with the ribosome biogenesis pathway and the general transcriptional profile of Chironomus riparius aquatic larvae were investigated by means of changes in the rDNA activity (through the study of the internal transcribed spacer 2, ITS2) and variations in the expression profile of ribosomal protein genes (rpL4, rpL11, and rpL13) after acute 24-h and 48-h exposures to a wide range of BBP doses. Furthermore, cytogenetic assays were conducted to evaluate the transcriptional activity of polytene chromosomes from salivary gland cells, with special attention to the nucleolus and the Balbiani rings (BRs) of chromosome IV. BBP caused a dose and time-dependent toxicity in most of the selected biomarkers, with a general depletion in the gene expression levels and the activity of BR2 after 48-h treatments. At the same time, decondensation and activation of some centromeres took place, while the activity of nucleolus remained unaltered. Withdrawal of the xenobiotic allowed the larvae to reach control levels in the case of rpL4 and rpL13 genes, which were previously slightly downregulated in 24-h tests. These data provide the first evidence on the interaction of BBP with the ribosome synthesis pathways, which results in a significant impairment of the functional activity of ribosomal protein genes. Thus, the depletion of ribosomes would be a long-term effect of BBP-induced cellular damage. These findings may have important implications for understanding the adverse biological effects of BBP in C. riparius, since they provide new sensitive biomarkers of BBP exposure and highlight the suitability of this organism for ecotoxicological risk assessment, especially in aquatic

  7. Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats

    PubMed Central

    Li, Lingyu; Xiao, Lina; Hou, Yanghao; He, Qi; Zhu, Jin; Li, Yixin; Wu, Jingxian; Zhao, Jing; Yu, Shanshan; Zhao, Yong

    2016-01-01

    Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α (AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/PGC-1α signaling pathway. To be able to test this, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h with Sesn2 silencing. At 24 h after reperfusion, we found that neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were greater in the Sesn2 siRNA group than in the MCAO group. To explore protective mechanisms, an AMPK activator was used. Expression levels of Sesn2, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2, and UCP2 were significantly increased following cerebral I/R. However, upregulation of these proteins was prevented by Sesn2 small interfering RNA (siRNA). In contrast, activation of AMPK with 5′-aminoimidazole-4-carboxamide riboside weakened the effects of Sesn2 siRNA. These results suggest that Sesn2 silencing may suppress mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1α pathway. PMID:27453548

  8. Fast-Suppressor Screening for New Components in Protein Trafficking, Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

    PubMed Central

    Zhao, Qiong; Gao, Caiji; Lee, PoShing; Liu, Lin; Li, Shaofang; Hu, Tangjin; Shen, Jinbo; Pan, Shuying; Ye, Hao; Chen, Yunru; Cao, Wenhan; Cui, Yong; Zeng, Peng; Yu, Sheng; Gao, Yangbin; Chen, Liang; Mo, Beixin; Liu, Xin; Xiao, Shi; Zhao, Yunde; Zhong, Silin; Chen, Xuemei; Jiang, Liwen

    2015-01-01

    Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants. PMID:26165498

  9. A single RNA-dependent RNA polymerase assembles with mutually exclusive nucleotidyl transferase subunits to direct different pathways of small RNA biogenesis.

    PubMed

    Lee, Suzanne Rebecca; Talsky, Kristin Benjamin; Collins, Kathleen

    2009-07-01

    Members of the conserved family of eukaryotic RNA-dependent RNA polymerases (Rdrs) synthesize double-stranded RNA (dsRNA) intermediates in diverse pathways of small RNA (sRNA) biogenesis and RNA-mediated silencing. Rdr-dependent pathways of sRNA production are poorly characterized relative to Rdr-independent pathways, and the Rdr enzymes themselves are poorly characterized relative to their viral RNA-dependent RNA polymerase counterparts. We previously described a physical and functional coupling of the Tetrahymena thermophila Rdr, Rdr1, and a Dicer enzyme, Dcr2, in the production of approximately 24-nucleotide (nt) sRNA in vitro. Here we characterize the endogenous complexes that harbor Rdr1, termed RDRCs. Distinct RDRCs assemble to contain Rdr1 and subsets of the total of four tightly Rdr1-associated proteins. Of particular interest are two RDRC subunits, Rdn1 and Rdn2, which possess noncanonical ribonucleotidyl transferase motifs. We show that the two Rdn proteins are uridine-specific polymerases of separate RDRCs. Two additional RDRC subunits, Rdf1 and Rdf2, are present only in RDRCs containing Rdn1. Rdr1 catalytic activity is retained in RDRCs purified from cell extracts lacking any of the nonessential RDRC subunits (Rdn2, Rdf1, Rdf2) or if the RDRC harbors a catalytically inactive Rdn. However, specific disruption of each RDRC imposes distinct loss-of-function consequences at the cellular level and has a differential impact on the accumulation of specific 23-24-nt sRNA sequences in vivo. The biochemical and biological phenotypes of RDRC subunit disruption reveal a previously unanticipated complexity of Rdr-dependent sRNA biogenesis in vivo. PMID:19451546

  10. 4-Hydroxyisoleucine improves insulin resistance by promoting mitochondrial biogenesis and act through AMPK and Akt dependent pathway.

    PubMed

    Rawat, Arun Kumar; Korthikunta, Venkateswarlu; Gautam, Sudeep; Pal, Savita; Tadigoppula, Narender; Tamrakar, Akhilesh Kumar; Srivastava, Arvind Kumar

    2014-12-01

    4-Hydroxyisoleucine (4-HIL) is an unusual amino acid isolated from fenugreek seeds (Trigonella foenum graecum L). Various studies have shown that it acts as an antidiabetic agent yet its mechanism of action is not clear. We therefore investigated the effect 4-HIL on the high fructose diet fed streptozotocin induced diabetic rats and L6 myotubes. 4-HIL (50 mg/kg) has improved blood lipid profile, glucose tolerance and insulin sensitivity in a diabetic rat model. It has increased the glucose uptake in L6 myotubes in AMPK-dependent manner and upregulated the expression of genes (PGC-1α, PGC-1β, CPT 1 and CPT 2), which have role in mitochondrial biogenesis and energy metabolism in the liver, skeletal muscles as well as in L6 myotubes. Interestingly, it also increased the AMPK and Akt expression along with their phosphorylated forms in the liver and muscle tissues of treated animals. Altogether we concluded that 4-HIL acts to improve insulin resistance by promoting mitochondrial biogenesis in high fructose diet fed STZ induced diabetic rats. PMID:25454462

  11. Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

    PubMed

    Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Verma, Deepika; Priyanka, Kumari; Bal, Amanjit; Gill, Kiran Dip

    2015-12-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases. PMID:26493151

  12. The impact of aging on mitochondrial function and biogenesis pathways in skeletal muscle of sedentary high- and low-functioning elderly individuals.

    PubMed

    Joseph, Anna-Maria; Adhihetty, Peter J; Buford, Thomas W; Wohlgemuth, Stephanie E; Lees, Hazel A; Nguyen, Linda M-D; Aranda, Juan M; Sandesara, Bhanu D; Pahor, Marco; Manini, Todd M; Marzetti, Emanuele; Leeuwenburgh, Christiaan

    2012-10-01

    Age-related loss of muscle mass and strength (sarcopenia) leads to a decline in physical function and frailty in the elderly. Among the many proposed underlying causes of sarcopenia, mitochondrial dysfunction is inherent in a variety of aged tissues. The intent of this study was to examine the effect of aging on key groups of regulatory proteins involved in mitochondrial biogenesis and how this relates to physical performance in two groups of sedentary elderly participants, classified as high- and low-functioning based on the Short Physical Performance Battery test. Muscle mass was decreased by 38% and 30% in low-functioning elderly (LFE) participants when compared to young and high-functioning elderly participants, respectively, and positively correlated to physical performance. Mitochondrial respiration in permeabilized muscle fibers was reduced (41%) in the LFE group when compared to the young, and this was associated with a 30% decline in cytochrome c oxidase activity. Levels of key metabolic regulators, SIRT3 and PGC-1α, were significantly reduced (50%) in both groups of elderly participants when compared to young. Similarly, the fusion protein OPA1 was lower in muscle from elderly subjects; however, no changes were detected in Mfn2, Drp1 or Fis1 among the groups. In contrast, protein import machinery components Tom22 and cHsp70 were increased in the LFE group when compared to the young. This study suggests that aging in skeletal muscle is associated with impaired mitochondrial function and altered biogenesis pathways and that this may contribute to muscle atrophy and the decline in muscle performance observed in the elderly population. PMID:22681576

  13. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

    PubMed Central

    López de Armentia, María Milagros; Amaya, Celina; Colombo, María Isabel

    2016-01-01

    Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis) or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila). The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII) are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections. PMID:27005665

  14. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles.

    PubMed

    López de Armentia, María Milagros; Amaya, Celina; Colombo, María Isabel

    2016-01-01

    Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis) or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila). The bacteria described in this review often use secretion systems to control the host's response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII) are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections. PMID:27005665

  15. Mitochondrial biogenesis: pharmacological approaches.

    PubMed

    Valero, Teresa

    2014-01-01

    ), myoclonic epilepsy with ragged-red fibers (MERRF), mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), Leber's hereditary optic neuropathy (LHON), the syndrome of neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP), and Leigh's syndrome. Likewise, other diseases in which mitochondrial dysfunction plays a very important role include neurodegenerative diseases, diabetes or cancer. Generally, in mitochondrial diseases a mutation in the mitochondrial DNA leads to a loss of functionality of the OXPHOS system and thus to a depletion of ATP and overproduction of ROS, which can, in turn, induce further mtDNA mutations. The work by Yu-Ting Wu, Shi-Bei Wu, and Yau-Huei Wei (Department of Biochemistry and Molecular Biology, National Yang-Ming University, Taiwan) [4] focuses on the aforementioned mitochondrial diseases with special attention to the compensatory mechanisms that prompt mitochondria to produce more energy even under mitochondrial defect-conditions. These compensatory mechanisms include the overexpression of antioxidant enzymes, mitochondrial biogenesis and overexpression of respiratory complex subunits, as well as metabolic shift to glycolysis. The pathways observed to be related to mitochondrial biogenesis as a compensatory adaptation to the energetic deficits in mitochondrial diseases are described (PGC- 1, Sirtuins, AMPK). Several pharmacological strategies to trigger these signaling cascades, according to these authors, are the use of bezafibrate to activate the PPAR-PGC-1α axis, the activation of AMPK by resveratrol and the use of Sirt1 agonists such as quercetin or resveratrol. Other strategies currently used include the addition of antioxidant supplements to the diet (dietary supplementation with antioxidants) such as L-carnitine, coenzyme Q10,MitoQ10 and other mitochondria-targeted antioxidants,N-acetylcysteine (NAC), vitamin C, vitamin E vitamin K1, vitamin B, sodium pyruvate or -lipoic acid. As aforementioned, other

  16. Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

    PubMed

    Krimmer, T; Rapaport, D; Ryan, M T; Meisinger, C; Kassenbrock, C K; Blachly-Dyson, E; Forte, M; Douglas, M G; Neupert, W; Nargang, F E; Pfanner, N

    2001-01-22

    Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore. PMID:11266446

  17. DEMONSTRATION BULLETIN: BIOGENESIS SOIL WASHING TECHNOLOGY - BIOGENESIS

    EPA Science Inventory

    The BioGenesisSM soil washing technology was developed by BioGenesis Enterprises, Inc. to remove organic compounds from soil. The technology uses a proprietary solution (BioGenesisSM cleaner) to transfer organic compounds from the soil matrix to a liquid phase. BioGenesis claims...

  18. OM2, a Novel Oligomannuronate-Chromium(III) Complex, Promotes Mitochondrial Biogenesis and Lipid Metabolism in 3T3-L1 Adipocytes via the AMPK-PGC1α Pathway

    PubMed Central

    Hao, Jiejie; Hao, Cui; Zhang, Lijuan; Liu, Xin; Zhou, Xiaolin; Dun, Yunlou; Li, Haihua; Li, Guangsheng; Zhao, Xiaoliang; An, Yuanyuan; Liu, Jiankang; Yu, Guangli

    2015-01-01

    Background In our previous studies, we prepared novel oligomannuronate-chromium(III) complexes (OM2, OM4) from marine alginate, and found that these compounds sensitize insulin action better than oligomannuronate(OM), chromium, and metformin in C2C12 skeletal muscle cells. In the present study, we studied their effects on mitochondrial biogenesis, lipid metabolism, and the underlying molecular mechanisms in differentiated 3T3-L1 adipocytes. Methodology/Principal Findings We firstly used the pGL3-PGC1α and pGL3-ATGL promoter plasmids to compare their effects on PGC1α and ATGL transcription activities. Then mitochondrial biogenesis was quantified by transmission electron microscopy and MitoTracker staining. Mitochondrial oxygen consumption and fatty acid oxidation were measured by an oxygen biosensor system and ³H-labelled water scintillation. The mitochondrial DNA and mRNA involved in mitochondrial biogenesis and lipid oxidation were evaluated by real-time PCR. AMPK together with other protein expression levels were measured by western blotting. The inhibitor compound C and siRNA of PGC1α were used to inhibit the OM2-induced AMPK-PGC1α signaling pathway. And we found that OM2 stimulated AMPK-PGC1α pathway in the 3T3-L1 adipocytes, which were correlated with induced mitochondrial biogenesis, improved mitochondrial function, and reduced lipid accumulation by enhanced fatty acid β-oxidation and augmented ATGL protein expression. Conclusions/Significance Our data indicated that the marine oligosaccharide-derived OM2 might represent a novel class of molecules that could be useful for type 2 diabetes prevention and treatment by up-regulating AMPK-PGC1α signaling pathway. PMID:26176781

  19. cAMP-induced Mitochondrial Compartment Biogenesis

    PubMed Central

    Yoboue, Edgar D.; Augier, Eric; Galinier, Anne; Blancard, Corinne; Pinson, Benoît; Casteilla, Louis; Rigoulet, Michel; Devin, Anne

    2012-01-01

    Cell fate and proliferation are tightly linked to the regulation of the mitochondrial energy metabolism. Hence, mitochondrial biogenesis regulation, a complex process that requires a tight coordination in the expression of the nuclear and mitochondrial genomes, has a major impact on cell fate and is of high importance. Here, we studied the molecular mechanisms involved in the regulation of mitochondrial biogenesis through a nutrient-sensing pathway, the Ras-cAMP pathway. Activation of this pathway induces a decrease in the cellular phosphate potential that alleviates the redox pressure on the mitochondrial respiratory chain. One of the cellular consequences of this modulation of cellular phosphate potential is an increase in the cellular glutathione redox state. The redox state of the glutathione disulfide-glutathione couple is a well known important indicator of the cellular redox environment, which is itself tightly linked to mitochondrial activity, mitochondria being the main cellular producer of reactive oxygen species. The master regulator of mitochondrial biogenesis in yeast (i.e. the transcriptional co-activator Hap4p) is positively regulated by the cellular glutathione redox state. Using a strain that is unable to modulate its glutathione redox state (Δglr1), we pinpoint a positive feedback loop between this redox state and the control of mitochondrial biogenesis. This is the first time that control of mitochondrial biogenesis through glutathione redox state has been shown. PMID:22396541

  20. Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation*

    PubMed Central

    Fernández-Pevida, Antonio; Rodríguez-Galán, Olga; Díaz-Quintana, Antonio; Kressler, Dieter; de la Cruz, Jesús

    2012-01-01

    Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process. PMID:22995916

  1. PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway

    PubMed Central

    Yang, Zhaolin; Chen, Kuan-Ming; Pandey, Radha Raman; Homolka, David; Reuter, Michael; Janeiro, Bruno Kotska Rodino; Sachidanandam, Ravi; Fauvarque, Marie-Odile; McCarthy, Andrew A.; Pillai, Ramesh S.

    2016-01-01

    Summary PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5′→3′ processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm. PMID:26669262

  2. Concerted removal of the Erb1-Ytm1 complex in ribosome biogenesis relies on an elaborate interface.

    PubMed

    Thoms, Matthias; Ahmed, Yasar Luqman; Maddi, Karthik; Hurt, Ed; Sinning, Irmgard

    2016-01-29

    The complicated process of eukaryotic ribosome biogenesis involves about 200 assembly factors that transiently associate with the nascent pre-ribosome in a spatiotemporally ordered way. During the early steps of 60S subunit formation, several proteins, collectively called A3 cluster factors, participate in the removal of the internal transcribed spacer 1 (ITS1) from 27SA3 pre-rRNA. Among these factors is the conserved hetero-trimeric Nop7-Erb1-Ytm1 complex (or human Pes1-Bop1-Wdr12), which is removed from the evolving pre-60S particle by the AAA ATPase Rea1 to allow progression in the pathway. Here, we clarify how Ytm1 and Erb1 interact, which has implications for the release mechanism of both factors from the pre-ribosome. Biochemical studies show that Ytm1 and Erb1 bind each other via their ß-propeller domains. The crystal structure of the Erb1-Ytm1 heterodimer determined at 2.67Å resolution reveals an extended interaction surface between the propellers in a rarely observed binding mode. Structure-based mutations in the interface that impair the Erb1-Ytm1 interaction do not support growth, with specific defects in 60S subunit synthesis. Under these mutant conditions, it becomes clear that an intact Erb1-Ytm1 complex is required for 60S maturation and that loss of this stable interaction prevents ribosome production. PMID:26657628

  3. [About the ribosomal biogenesis in human].

    PubMed

    Tafforeau, Lionel

    2015-01-01

    Ribosomes are cellular ribonucleoprotein particles required for a fundamental mechanism, translation of the genetic information into proteins. Ribosome biogenesis is a highly complex pathway involving many maturation steps: ribosomal RNA (rRNA) synthesis, rRNA processing, pre-rRNA modifications, its assembly with ribosomal proteins in the nuceolus, export of the subunit precursors to the nucleoplasm and the cytoplasm. Ribosome biogenesis has mainly being investigated in yeast during these last 25 years. However, recent works have shown that, despite many similarities between yeast and human ribosome structure and biogenesis, human pre-rRNA processing is far more complex than in yeast. In order to better understand diseases related to a malfunction in ribosome synthesis, the ribosomopathies, research should be conducted directly in human cells and animal models. PMID:26152166

  4. TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

    PubMed Central

    Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.

    2013-01-01

    Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance. PMID:23171550

  5. Plant Peroxisomes: Biogenesis and Function

    PubMed Central

    Hu, Jianping; Baker, Alison; Bartel, Bonnie; Linka, Nicole; Mullen, Robert T.; Reumann, Sigrun; Zolman, Bethany K.

    2012-01-01

    Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle’s dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense. PMID:22669882

  6. The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly.

    PubMed

    Tutuncuoglu, Beril; Jakovljevic, Jelena; Wu, Shan; Gao, Ning; Woolford, John L

    2016-09-01

    Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo-that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling. PMID:27390266

  7. Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.

    PubMed

    Wu, Shan; Tutuncuoglu, Beril; Yan, Kaige; Brown, Hailey; Zhang, Yixiao; Tan, Dan; Gamalinda, Michael; Yuan, Yi; Li, Zhifei; Jakovljevic, Jelena; Ma, Chengying; Lei, Jianlin; Dong, Meng-Qiu; Woolford, John L; Gao, Ning

    2016-06-01

    Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly. PMID:27251291

  8. The effects of heat induction and the siRNA biogenesis pathway on the transgenerational transposition of ONSEN, a copia-like retrotransposon in Arabidopsis thaliana.

    PubMed

    Matsunaga, Wataru; Kobayashi, Akie; Kato, Atsushi; Ito, Hidetaka

    2012-05-01

    Environmental stress influences genetic and epigenetic regulation in plant genomes. We previously reported that heat stress activated a copia-like retrotransposon named ONSEN. To investigate the heat sensitivity and transgenerational activation of ONSEN, we analyzed the stress response by temperature shift and multiple heat stress treatments. ONSEN was activated at 37°C, and the newly inserted ONSEN was transcriptionally active and mobile to the next generation subjected to heat stress, indicating that the regulation of ONSEN is independent of positional effects on the chromosome. Reciprocal crosses with activated ONSEN revealed that the transgenerational transposition was inherited from both sexes, indicating that the transposition is suppressed independently of gametophytic regulation. We showed previously that ONSEN was transposed in mutants deficient in small interfering RNA (siRNA) biogenesis, including nrpd2 and rdr2, but not dcl3. To define the functional redundancy of Dicer-like (DCL) proteins in Arabidopsis, we analyzed ONSEN activation in mutants deficient in DCL proteins, including dcl2, dcl3 and dcl4. ONSEN was nearly immobile in a single Dicer mutant; however, some transgenerational transpositions were observed in dcl2/dcl3/dcl4 triple mutants subjected to heat stress. This indicated that the Dicer family is redundant for ONSEN transposition. To examine the activation of ONSEN in undifferentiated cells, ONSEN transcripts and synthesized DNA were analyzed in heat-stressed callus tissue. In contrast to vegetative tissue, high accumulation of the transcripts and amplified DNA copies of ONSEN were detected in callus. This result indicated that ONSEN activation is controlled by cell-specific regulatory mechanisms. PMID:22173101

  9. DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation.

    PubMed

    Tummala, Hemanth; Walne, Amanda J; Williams, Mike; Bockett, Nicholas; Collopy, Laura; Cardoso, Shirleny; Ellison, Alicia; Wynn, Rob; Leblanc, Thierry; Fitzgibbon, Jude; Kelsell, David P; van Heel, David A; Payne, Elspeth; Plagnol, Vincent; Dokal, Inderjeet; Vulliamy, Tom

    2016-07-01

    A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit. PMID:27346687

  10. Biogenesis of light harvesting proteins.

    PubMed

    Dall'Osto, Luca; Bressan, Mauro; Bassi, Roberto

    2015-09-01

    The LHC family includes nuclear-encoded, integral thylakoid membrane proteins, most of which coordinate chlorophyll and xanthophyll chromophores. By assembling with the core complexes of both photosystems, LHCs form a flexible peripheral moiety for enhancing light-harvesting cross-section, regulating its efficiency and providing protection against photo-oxidative stress. Upon its first appearance, LHC proteins underwent evolutionary diversification into a large protein family with a complex genetic redundancy. Such differentiation appears as a crucial event in the adaptation of photosynthetic organisms to changing environmental conditions and land colonization. The structure of photosystems, including nuclear- and chloroplast-encoded subunits, presented the cell with a number of challenges for the control of the light harvesting function. Indeed, LHC-encoding messages are translated in the cytosol, and pre-proteins imported into the chloroplast, processed to their mature size and targeted to the thylakoids where are assembled with chromophores. Thus, a tight coordination between nuclear and plastid gene expression, in response to environmental stimuli, is required to adjust LHC composition during photoacclimation. In recent years, remarkable progress has been achieved in elucidating structure, function and regulatory pathways involving LHCs; however, a number of molecular details still await elucidation. In this review, we will provide an overview on the current knowledge on LHC biogenesis, ranging from organization of pigment-protein complexes to the modulation of gene expression, import and targeting to the photosynthetic membranes, and regulation of LHC assembly and turnover. Genes controlling these events are potential candidate for biotechnological applications aimed at optimizing light use efficiency of photosynthetic organisms. This article is part of a Special Issue entitled: Chloroplast biogenesis. PMID:25687893

  11. Human disorders of peroxisome metabolism and biogenesis.

    PubMed

    Waterham, Hans R; Ferdinandusse, Sacha; Wanders, Ronald J A

    2016-05-01

    Peroxisomes are dynamic organelles that play an essential role in a variety of cellular catabolic and anabolic metabolic pathways, including fatty acid alpha- and beta-oxidation, and plasmalogen and bile acid synthesis. Defects in genes encoding peroxisomal proteins can result in a large variety of peroxisomal disorders either affecting specific metabolic pathways, i.e., the single peroxisomal enzyme deficiencies, or causing a generalized defect in function and assembly of peroxisomes, i.e., peroxisome biogenesis disorders. In this review, we discuss the clinical, biochemical, and genetic aspects of all human peroxisomal disorders currently known. PMID:26611709

  12. Concerted removal of the Erb1–Ytm1 complex in ribosome biogenesis relies on an elaborate interface

    PubMed Central

    Thoms, Matthias; Ahmed, Yasar Luqman; Maddi, Karthik; Hurt, Ed; Sinning, Irmgard

    2016-01-01

    The complicated process of eukaryotic ribosome biogenesis involves about 200 assembly factors that transiently associate with the nascent pre-ribosome in a spatiotemporally ordered way. During the early steps of 60S subunit formation, several proteins, collectively called A3 cluster factors, participate in the removal of the internal transcribed spacer 1 (ITS1) from 27SA3 pre-rRNA. Among these factors is the conserved hetero-trimeric Nop7–Erb1–Ytm1 complex (or human Pes1–Bop1–Wdr12), which is removed from the evolving pre-60S particle by the AAA ATPase Rea1 to allow progression in the pathway. Here, we clarify how Ytm1 and Erb1 interact, which has implications for the release mechanism of both factors from the pre-ribosome. Biochemical studies show that Ytm1 and Erb1 bind each other via their ß-propeller domains. The crystal structure of the Erb1–Ytm1 heterodimer determined at 2.67Å resolution reveals an extended interaction surface between the propellers in a rarely observed binding mode. Structure-based mutations in the interface that impair the Erb1–Ytm1 interaction do not support growth, with specific defects in 60S subunit synthesis. Under these mutant conditions, it becomes clear that an intact Erb1–Ytm1 complex is required for 60S maturation and that loss of this stable interaction prevents ribosome production. PMID:26657628

  13. MYC and Mitochondrial Biogenesis

    PubMed Central

    Morrish, Fionnuala; Hockenbery, David

    2014-01-01

    Mitochondria, the powerhouses of the cell, face two imperatives concerning biogenesis. The first is the requirement for dividing cells to replicate their mitochondrial content by growth of existing mitochondria. The second is the dynamic regulation of mitochondrial content in response to organismal and cellular cues (e.g., exercise, caloric restriction, energy status, temperature). MYC provides the clearest example of a programmed expansion of mitochondrial content linked to the cell cycle. As an oncogene, MYC also presents intriguing questions about the role of its mitochondrial targets in cancer-related phenotypes, such as the Warburg effect and MYC-dependent apoptosis. PMID:24789872

  14. Peroxisome Biogenesis and Function

    PubMed Central

    Kaur, Navneet; Reumann, Sigrun; Hu, Jianping

    2009-01-01

    Peroxisomes are small and single membrane-delimited organelles that execute numerous metabolic reactions and have pivotal roles in plant growth and development. In recent years, forward and reverse genetic studies along with biochemical and cell biological analyses in Arabidopsis have enabled researchers to identify many peroxisome proteins and elucidate their functions. This review focuses on the advances in our understanding of peroxisome biogenesis and metabolism, and further explores the contribution of large-scale analysis, such as in sillco predictions and proteomics, in augmenting our knowledge of peroxisome function In Arabidopsis. PMID:22303249

  15. Curli Biogenesis and Function

    PubMed Central

    Barnhart, Michelle M.; Chapman, Matthew R.

    2010-01-01

    Curli are the major proteinaceous component of a complex extra-cellular matrix produced by many Enterobacteriaceae. Curli were first discovered in the late 1980s on Escherichia coli strains that caused bovine mastitis, and have since been implicated in many physiological and pathogenic processes of E. coli and Salmonella spp. Curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation. Curli also mediate host cell adhesion and invasion, and they are potent inducers of the host inflammatory response. The structure and biogenesis of curli are unique among bacterial fibers that have been described to date. Structurally and biochemically, curli belong to a growing class of fibers known as amyloids. Amyloid fiber formation is responsible for several human diseases including Alzheimer's, Huntington's, and prion diseases, although the process of in vivo amyloid formation is not well understood. Curli provide a unique system to study macromolecular assembly in bacteria and in vivo amyloid fiber formation. Here, we review curli biogenesis, regulation, role in biofilm formation, and role in pathogenesis. PMID:16704339

  16. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

    PubMed

    Costello, Shawn M; Plummer, Ashlee M; Fleming, Patrick J; Fleming, Karen G

    2016-08-16

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

  17. Poxvirus Membrane Biogenesis

    PubMed Central

    2015-01-01

    Poxviruses differ from most DNA viruses by replicating entirely within the cytoplasm. The first discernible viral structures are crescents and spherical immature virions containing a single lipoprotein membrane bilayer with an external honeycomb lattice. Because this viral membrane displays no obvious continuity with a cellular organelle, a de novo origin was suggested. Nevertheless, transient connections between viral and cellular membranes could be difficult to resolve. Despite the absence of direct evidence, the intermediate compartment (ERGIC) between the endoplasmic reticulum (ER) and Golgi apparatus and the ER itself were considered possible sources of crescent membranes. A break-through in understanding poxvirus membrane biogenesis has come from recent studies of the abortive replication of several vaccinia virus null mutants. Novel images showing continuity between viral crescents and the ER and the accumulation of immature virions in the expanded ER lumen provide the first direct evidence for a cellular origin of this poxvirus membrane. PMID:25728299

  18. Poxvirus membrane biogenesis.

    PubMed

    Moss, Bernard

    2015-05-01

    Poxviruses differ from most DNA viruses by replicating entirely within the cytoplasm. The first discernible viral structures are crescents and spherical immature virions containing a single lipoprotein membrane bilayer with an external honeycomb lattice. Because this viral membrane displays no obvious continuity with a cellular organelle, a de novo origin was suggested. Nevertheless, transient connections between viral and cellular membranes could be difficult to resolve. Despite the absence of direct evidence, the intermediate compartment (ERGIC) between the endoplasmic reticulum (ER) and Golgi apparatus and the ER itself were considered possible sources of crescent membranes. A break-through in understanding poxvirus membrane biogenesis has come from recent studies of the abortive replication of several vaccinia virus null mutants. Novel images showing continuity between viral crescents and the ER and the accumulation of immature virions in the expanded ER lumen provide the first direct evidence for a cellular origin of this poxvirus membrane. PMID:25728299

  19. Unravelling the mechanisms regulating muscle mitochondrial biogenesis.

    PubMed

    Hood, David A; Tryon, Liam D; Carter, Heather N; Kim, Yuho; Chen, Chris C W

    2016-08-01

    Skeletal muscle is a tissue with a low mitochondrial content under basal conditions, but it is responsive to acute increases in contractile activity patterns (i.e. exercise) which initiate the signalling of a compensatory response, leading to the biogenesis of mitochondria and improved organelle function. Exercise also promotes the degradation of poorly functioning mitochondria (i.e. mitophagy), thereby accelerating mitochondrial turnover, and preserving a pool of healthy organelles. In contrast, muscle disuse, as well as the aging process, are associated with reduced mitochondrial quality and quantity in muscle. This has strong negative implications for whole-body metabolic health and the preservation of muscle mass. A number of traditional, as well as novel regulatory pathways exist in muscle that control both biogenesis and mitophagy. Interestingly, although the ablation of single regulatory transcription factors within these pathways often leads to a reduction in the basal mitochondrial content of muscle, this can invariably be overcome with exercise, signifying that exercise activates a multitude of pathways which can respond to restore mitochondrial health. This knowledge, along with growing realization that pharmacological agents can also promote mitochondrial health independently of exercise, leads to an optimistic outlook in which the maintenance of mitochondrial and whole-body metabolic health can be achieved by taking advantage of the broad benefits of exercise, along with the potential specificity of drug action. PMID:27470593

  20. Germ plasm biogenesis –an Oskar-centric perspective

    PubMed Central

    Lehmann, Ruth

    2016-01-01

    Germ granules are the hallmark of all germ cells. These membrane-less, electron-dense structures were first observed over 100 years ago. Today, their role in regulating and processing transcripts critical for the establishment, maintenance and protection of germ cells is well-established and pathways outlining the biochemical mechanisms and physical properties associated with their biogenesis are emerging. PMID:26970648

  1. The structure of Rpf2-Rrs1 explains its role in ribosome biogenesis.

    PubMed

    Kharde, Satyavati; Calviño, Fabiola R; Gumiero, Andrea; Wild, Klemens; Sinning, Irmgard

    2015-08-18

    The assembly of eukaryotic ribosomes is a hierarchical process involving about 200 biogenesis factors and a series of remodeling steps. The 5S RNP consisting of the 5S rRNA, RpL5 and RpL11 is recruited at an early stage, but has to rearrange during maturation of the pre-60S ribosomal subunit. Rpf2 and Rrs1 have been implicated in 5S RNP biogenesis, but their precise role was unclear. Here, we present the crystal structure of the Rpf2-Rrs1 complex from Aspergillus nidulans at 1.5 Å resolution and describe it as Brix domain of Rpf2 completed by Rrs1 to form two anticodon-binding domains with functionally important tails. Fitting the X-ray structure into the cryo-EM density of a previously described pre-60S particle correlates with biochemical data. The heterodimer forms specific contacts with the 5S rRNA, RpL5 and the biogenesis factor Rsa4. The flexible protein tails of Rpf2-Rrs1 localize to the central protuberance. Two helices in the Rrs1 C-terminal tail occupy a strategic position to block the rotation of 25S rRNA and the 5S RNP. Our data provide a structural model for 5S RNP recruitment to the pre-60S particle and explain why removal of Rpf2-Rrs1 is necessary for rearrangements to drive 60S maturation. PMID:26117542

  2. Aβ25-35 Suppresses Mitochondrial Biogenesis in Primary Hippocampal Neurons.

    PubMed

    Dong, Weiguo; Wang, Feng; Guo, Wanqing; Zheng, Xuehua; Chen, Yue; Zhang, Wenguang; Shi, Hong

    2016-01-01

    Mitochondrial biogenesis is involved in the regulation of mitochondrial content, morphology, and function. Impaired mitochondrial biogenesis has been observed in Alzheimer's disease. Amyloid-β (Aβ) has been shown to cause mitochondrial dysfunction in cultured neurons, but its role in mitochondrial biogenesis in neurons remains poorly defined. AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) are key energy-sensing molecules regulating mitochondrial biogenesis. In addition, peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis, is a target for SIRT1 deacetylase activity. In this study, we investigated the effects of Aβ25-35 on mitochondrial biogenesis in cultured hippocampal neurons and the underlying mechanisms. In primary hippocampal neurons, we found that 24-h incubation with Aβ25-35 suppressed both phosphorylations of AMPK and SIRT1 expression and increased PGC-1α acetylation expression. In addition, Aβ25-35 also resulted in a decrease in mitochondrial DNA copy number, as well as decreases in the expression of mitochondrial biogenesis factors (PGC-1α, NRF 1, NRF 2, and Tfam). Taken together, these data show that Aβ25-35 suppresses mitochondrial biogenesis in hippocampal neurons. Aβ25-35-induced impairment of mitochondrial biogenesis may be associated with the inhibition of the AMPK-SIRT1-PGC-1α pathway. PMID:26055049

  3. Peroxisomal Biogenesis in Ischemic Brain

    PubMed Central

    Young, Jennifer M.; Nelson, Jonathan W.; Cheng, Jian; Zhang, Wenri; Mader, Sarah; Davis, Catherine M.; Morrison, Richard S.

    2015-01-01

    Abstract Aims: Peroxisomes are highly adaptable and dynamic organelles, adjusting their size, number, and enzyme composition to changing environmental and metabolic demands. We determined whether peroxisomes respond to ischemia, and whether peroxisomal biogenesis is an adaptive response to cerebral ischemia. Results: Focal cerebral ischemia induced peroxisomal biogenesis in peri-infarct neurons, which was associated with a corresponding increase in peroxisomal antioxidant enzyme catalase. Peroxisomal biogenesis was also observed in primary cultured cortical neurons subjected to ischemic insult induced by oxygen-glucose deprivation (OGD). A catalase inhibitor increased OGD-induced neuronal death. Moreover, preventing peroxisomal proliferation by knocking down dynamin-related protein 1 (Drp1) exacerbated neuronal death induced by OGD, whereas enhancing peroxisomal biogenesis pharmacologically using a peroxisome proliferator-activated receptor-alpha agonist protected against neuronal death induced by OGD. Innovation: This is the first documentation of ischemia-induced peroxisomal biogenesis in mammalian brain using a combined in vivo and in vitro approach, electron microscopy, high-resolution laser-scanning confocal microscopy, and super-resolution structured illumination microscopy. Conclusion: Our findings suggest that neurons respond to ischemic injury by increasing peroxisome biogenesis, which serves a protective function, likely mediated by enhanced antioxidant capacity of neurons. Antioxid. Redox Signal. 22, 109–120. PMID:25226217

  4. Mitochondrial biogenesis is required for axonal growth.

    PubMed

    Vaarmann, Annika; Mandel, Merle; Zeb, Akbar; Wareski, Przemyslaw; Liiv, Joanna; Kuum, Malle; Antsov, Eva; Liiv, Mailis; Cagalinec, Michal; Choubey, Vinay; Kaasik, Allen

    2016-06-01

    During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKβ, LKB1-STRAD or TAK1 pathways also co-activate the AMPK-PGC-1α-NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized. PMID:27122166

  5. Minotaur is critical for primary piRNA biogenesis

    PubMed Central

    Vagin, Vasily V.; Yu, Yang; Jankowska, Anna; Luo, Yicheng; Wasik, Kaja A.; Malone, Colin D.; Harrison, Emily; Rosebrock, Adam; Wakimoto, Barbara T.; Fagegaltier, Delphine; Muerdter, Felix; Hannon, Gregory J.

    2013-01-01

    Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur. PMID:23788724

  6. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon.

    PubMed

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S

    2015-06-15

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. PMID:25877867

  7. Biogenesis of Plant Prevacuolar Multivesicular Bodies.

    PubMed

    Cui, Yong; Shen, Jinbo; Gao, Caiji; Zhuang, Xiaohong; Wang, Junqi; Jiang, Liwen

    2016-06-01

    Plant prevacuolar compartments (PVCs), or multivesicular bodies (MVBs), are single membrane-bound organelles that play important roles in mediating protein trafficking to vacuoles in the secretory pathway. PVC/MVB also serves as a late endosome in the endocytic pathway in plants. Since the plant PVC was identified as an MVB more than 10 years ago, great progress has been made toward the understanding of PVC/MVB function and biogenesis in plants. In this review, we first summarize previous research into the identification and characterization of plant PVCs/MVBs, and then highlight recent advances on the mechanisms underlying intraluminal vesicle formation and maturation of plant PVCs/MVBs. In addition, we discuss the possible crosstalk that appears to occur between PVCs/MVBs and autophagosomes during autophagy in plants. Finally, we list some open questions and present future perspectives in this field. PMID:26836198

  8. RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

    PubMed Central

    Réty, Stéphane; Lebaron, Simon; Deschamps, Patrick; Bareille, Joseph; Jombart, Julie; Robert-Paganin, Julien; Delbos, Lila; Chardon, Florian; Zhang, Elodie; Charenton, Clément; Tollervey, David; Leulliot, Nicolas

    2014-01-01

    During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation. PMID:24823650

  9. Ribosome biogenesis: emerging evidence for a central role in the regulation of skeletal muscle mass†

    PubMed Central

    Chaillou, Thomas; Kirby, Tyler J.; McCarthy, John J.

    2016-01-01

    The ribosome is a supramolecular ribonucleoprotein complex that functions at the heart of the translation machinery to convert mRNA into protein. Ribosome biogenesis is the primary determinant of translational capacity of the cell and accordingly has an essential role in the control of cell growth in eukaryotes. Cumulative evidence supports the hypothesis that ribosome biogenesis has an important role in the regulation of skeletal muscle mass. The purpose of this review is to, first, summarize the main mechanisms known to regulate ribosome biogenesis and, second, put forth the hypothesis that ribosome biogenesis is a central mechanism used by skeletal muscle to regulate protein synthesis and control skeletal muscle mass in response to anabolic and catabolic stimuli. The mTORC1 and Wnt/β-catenin/c-myc signaling pathways are discussed as the major pathways that work in concert with each of the three RNA polymerases (RNA Pol I, II and III) in regulating ribosome biogenesis. Consistent with our hypothesis, activation of these two pathways has been shown to be associated with ribosome biogenesis during skeletal muscle hypertrophy. Although further study is required, the finding that ribosome biogenesis is altered under catabolic states, in particular during disuse atrophy, suggests that its activation represents a novel therapeutic target to reduce or prevent muscle atrophy. Lastly, the emerging field of ribosome specialization is discussed and its potential role in the regulation of gene expression during periods of skeletal muscle plasticity. PMID:24604615

  10. Magnetosome biogenesis in magnetotactic bacteria.

    PubMed

    Uebe, René; Schüler, Dirk

    2016-09-13

    Magnetotactic bacteria derive their magnetic orientation from magnetosomes, which are unique organelles that contain nanometre-sized crystals of magnetic iron minerals. Although these organelles have evident potential for exciting biotechnological applications, a lack of genetically tractable magnetotactic bacteria had hampered the development of such tools; however, in the past decade, genetic studies using two model Magnetospirillum species have revealed much about the mechanisms of magnetosome biogenesis. In this Review, we highlight these new insights and place the molecular mechanisms of magnetosome biogenesis in the context of the complex cell biology of Magnetospirillum spp. Furthermore, we discuss the diverse properties of magnetosome biogenesis in other species of magnetotactic bacteria and consider the value of genetically 'magnetizing' non-magnetotactic bacteria. Finally, we discuss future prospects for this highly interdisciplinary and rapidly advancing field. PMID:27620945

  11. Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors

    PubMed Central

    Zhang, Gao; Frederick, Dennie T.; Wu, Lawrence; Wei, Zhi; Krepler, Clemens; Srinivasan, Satish; Chae, Young Chan; Xu, Xiaowei; Choi, Harry; Dimwamwa, Elaida; Shannan, Batool; Basu, Devraj; Zhang, Dongmei; Guha, Manti; Xiao, Min; Randell, Sergio; Sproesser, Katrin; Xu, Wei; Liu, Jephrey; Karakousis, Giorgos C.; Schuchter, Lynn M.; Gangadhar, Tara C.; Amaravadi, Ravi K.; Gu, Mengnan; Xu, Caiyue; Ghosh, Abheek; Xu, Weiting; Tian, Tian; Zhang, Jie; Zha, Shijie; Brafford, Patricia; Weeraratna, Ashani; Davies, Michael A.; Wargo, Jennifer A.; Avadhani, Narayan G.; Lu, Yiling; Mills, Gordon B.; Altieri, Dario C.; Flaherty, Keith T.

    2016-01-01

    Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi. PMID:27043285

  12. Folding and Biogenesis of Mitochondrial Small Tim Proteins

    PubMed Central

    Ceh-Pavia, Efrain; Spiller, Michael P.; Lu, Hui

    2013-01-01

    Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS. PMID:23945562

  13. Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors.

    PubMed

    Zhang, Gao; Frederick, Dennie T; Wu, Lawrence; Wei, Zhi; Krepler, Clemens; Srinivasan, Satish; Chae, Young Chan; Xu, Xiaowei; Choi, Harry; Dimwamwa, Elaida; Ope, Omotayo; Shannan, Batool; Basu, Devraj; Zhang, Dongmei; Guha, Manti; Xiao, Min; Randell, Sergio; Sproesser, Katrin; Xu, Wei; Liu, Jephrey; Karakousis, Giorgos C; Schuchter, Lynn M; Gangadhar, Tara C; Amaravadi, Ravi K; Gu, Mengnan; Xu, Caiyue; Ghosh, Abheek; Xu, Weiting; Tian, Tian; Zhang, Jie; Zha, Shijie; Liu, Qin; Brafford, Patricia; Weeraratna, Ashani; Davies, Michael A; Wargo, Jennifer A; Avadhani, Narayan G; Lu, Yiling; Mills, Gordon B; Altieri, Dario C; Flaherty, Keith T; Herlyn, Meenhard

    2016-05-01

    Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi. PMID:27043285

  14. The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

    PubMed Central

    Sardana, Richa; Johnson, Arlen W.

    2012-01-01

    We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates. PMID:22956767

  15. Biogenesis of respiratory cytochromes in bacteria.

    PubMed Central

    Thöny-Meyer, L

    1997-01-01

    Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible. PMID:9293186

  16. Insulin Granule Biogenesis, Trafficking and Exocytosis

    PubMed Central

    Hou, June Chunqiu; Min, Le; Pessin, Jeffrey E.

    2015-01-01

    It is becoming increasingly apparent that beta cell dysfunction resulting in abnormal insulin secretion is the essential element in the progression of patients from a state of impaired glucose tolerance to frank type 2 diabetes (Del Prato, 2003; Del Prato and Tiengo, 2001). Although extensive studies have examined the molecular, cellular and physiologic mechanisms of insulin granule biogenesis, sorting, and exocytosis the precise mechanisms controlling these processes and their dysregulation in the developed of diabetes remains an area of important investigation. We now know that insulin biogenesis initiates with the synthesis of preproinsulin in rough endoplastic reticulum and conversion of preproinsulin to proinsulin. Proinsulin begins to be packaged in the Trans-Golgi Network and is sorting into immature secretory granules. These immature granules become acidic via ATP-dependent proton pump and proinsulin undergoes proteolytic cleavage resulting the formation of insulin and C-peptide. During the granule maturation process, insulin is crystallized with zinc and calcium in the form of dense-core granules and unwanted cargo and membrane proteins undergo selective retrograde trafficking to either the constitutive trafficking pathway for secretion or to degradative pathways. The newly formed mature dense-core insulin granules populate two different intracellular pools, the readily releasable pools (RRP) and the reserved pool. These two distinct populations are thought to be responsible for the biphasic nature of insulin release in which the RRP granules are associated with the plasma membrane and undergo an acute calcium-dependent release accounting for first phase insulin secretion. In contrast, second phase insulin secretion requires the trafficking of the reserved granule pool to the plasma membrane. The initial trigger for insulin granule fusion with the plasma membrane is a rise in intracellular calcium and in the case of glucose stimulation results from

  17. Activation of the tumor suppressor p53 upon impairment of ribosome biogenesis.

    PubMed

    Bursac, Sladana; Brdovcak, Maja Cokaric; Donati, Giulio; Volarevic, Sinisa

    2014-06-01

    Errors in ribosome biogenesis can result in quantitative or qualitative defects in protein synthesis and consequently lead to improper execution of the genetic program and the development of specific diseases. Evidence has accumulated over the last decade suggesting that perturbation of ribosome biogenesis triggers a p53-activating checkpoint signaling pathway, often referred to as the ribosome biogenesis stress checkpoint pathway. Although it was originally suggested that p53 has a prominent role in preventing diseases by monitoring the fidelity of ribosome biogenesis, recent work has demonstrated that p53 activation upon impairment of ribosome biogenesis also mediates pathological manifestations in humans. Perturbations of ribosome biogenesis can trigger a p53-dependent checkpoint signaling pathway independent of DNA damage and the tumor suppressor ARF through inhibitory interactions of specific ribosomal components with the p53 negative regulator, Mdm2. Here we review the recent advances made toward understanding of this newly-recognized checkpoint signaling pathway, its role in health and disease, and discuss possible future directions in this exciting research field. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. PMID:24514102

  18. Cellulose biogenesis in Dictyostelium discoideum

    SciTech Connect

    Blanton, R.L.

    1993-12-31

    Organisms that synthesize cellulose can be found amongst the bacteria, protistans, fungi, and animals, but it is in plants that the importance of cellulose in function (as the major structural constituent of plant cell walls) and economic use (as wood and fiber) can be best appreciated. The structure of cellulose and its biosynthesis have been the subjects of intense investigation. One of the most important insights gained from these studies is that the synthesis of cellulose by living organisms involves much more than simply the polymerization of glucose into a (1{r_arrow}4)-{beta}-linked polymer. The number of glucoses in a polymer (the degree of polymerization), the crystalline form assumed by the glucan chains when they crystallize to form a microfibril, and the dimensions and orientation of the microfibrils are all subject to cellular control. Instead of cellulose biosynthesis, a more appropriate term might be cellulose biogenesis, to emphasize the involvement of cellular structures and mechanisms in controlling polymerization and directing crystallization and deposition. Dictyostelium discoideum is uniquely suitable for the study of cellulose biogenesis because of its amenability to experimental study and manipulation and the extent of our knowledge of its basic cellular mechanisms (as will be evident from the rest of this volume). In this chapter, I will summarize what is known about cellulose biogenesis in D. discoideum, emphasizing its potential to illuminate our understanding both of D. discoideum development and plant cellulose biogenesis.

  19. The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis

    PubMed Central

    Jeon, Young; Park, Yong-Joon; Cho, Hui Kyung; Jung, Hyun Ju; Ahn, Tae-Kyu; Kang, Hunseung; Pai, Hyun-Sook

    2015-01-01

    Nucleostemin is a nucleolar GTP-binding protein that is involved in stem cell proliferation, embryonic development, and ribosome biogenesis in mammals. Plant nucleostemin-like 1 (NSN1) plays a role in embryogenesis, and apical and floral meristem development. In this study, a nucleolar function of NSN1 in the regulation of ribosome biogenesis was identified. Green fluorescent protein (GFP)-fused NSN1 localized to the nucleolus, which was primarily determined by its N-terminal domain. Recombinant NSN1 and its N-terminal domain (NSN1-N) bound to RNA in vitro. Recombinant NSN1 expressed GTPase activity in vitro. NSN1 silencing in Arabidopsis thaliana and Nicotiana benthamiana led to growth retardation and premature senescence. NSN1 interacted with Pescadillo and EBNA1 binding protein 2 (EBP2), which are nucleolar proteins involved in ribosome biogenesis, and with several ribosomal proteins. NSN1, NSN1-N, and EBP2 co-fractionated primarily with the 60S ribosomal large subunit in vivo. Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation. NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression. Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis. PMID:26163696

  20. Fe/S protein biogenesis in trypanosomes - A review.

    PubMed

    Lukeš, Julius; Basu, Somsuvro

    2015-06-01

    Trypanosoma brucei, the causative agent of the African sleeping sickness of humans, and other kinetoplastid flagellates belong to the eukarytotic supergroup Excavata. This early-branching model protist is known for a broad range of unique features. As it is amenable to most techniques of forward and reverse genetics, T. brucei was subject to several studies of its iron-sulfur (Fe/S) protein biogenesis and thus represents the best studied excavate eukaryote. Here we review what is known about the Fe/S protein biogenesis of T. brucei, and focus especially on the comparative and evolutionary interesting aspects. We also explore the connections between the well-known and quite conserved ISC and CIA machineries and the tRNA thiolation pathway. Moreover, the Fe/S cluster protein biogenesis is dissected in the procyclic stage of T. brucei which has an active mitochondrion, as well as in its pathogenic bloodstream stage with a metabolically repressed organelle. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. PMID:25196712

  1. Biogenesis of pipecolic acid in Rhizoctonia leguminicola

    SciTech Connect

    Wickwire, B.M.

    1989-01-01

    This laboratory has long been interested in the biogenesis and biological properties of two indolizidine alkaloids, slaframine and swainsonine that are produced by the fungal parasite Rhizoctonia Leguminicola. Slaframine, (1S,6S,8aS-1 acetoxy-6-aminooctahydroindolizine) is a parasympathetic secretagogue, and swainsonine (1S,2R,8R,8aR-1,2,8-trihydroxyoctahydroindolizine) is a potent {alpha}-mannosidase inhibitor. This thesis concerns the initial steps of the biosynthesis of these alkaloids from lysine, via the common intermediate pipecolic acid, in whole cells and cell free enzyme systems of R. leguminicola. In confirmation of earlier work performed in this laboratory, L-lysine was used preferentially for pipecolate biosynthesis in R. Leguminicola. This pathway was supported by the finding that cell free extracts of R. leguminicola consistently converted L-(U-{sup 14}C)-lysine to three labelled metabolites: saccharopine, peak II, and pipecolic acid. Peak II was subsequently identified by appropriate proton NMR studies to be {delta}{sup 1}-piperideine-6-carboxylate, and the following pathway of pipecolic acid formation was postulated: L-lysine {yields} saccharopine {yields} {delta}{sup 1}-piperideine-6-carboxylate {yields} pipecolate. This pathway was confirmed by demonstration of each enzymatic step in vitro from purified radiolabeled substrates.

  2. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    SciTech Connect

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  3. The structure of Rpf2–Rrs1 explains its role in ribosome biogenesis

    PubMed Central

    Kharde, Satyavati; Calviño, Fabiola R.; Gumiero, Andrea; Wild, Klemens; Sinning, Irmgard

    2015-01-01

    The assembly of eukaryotic ribosomes is a hierarchical process involving about 200 biogenesis factors and a series of remodeling steps. The 5S RNP consisting of the 5S rRNA, RpL5 and RpL11 is recruited at an early stage, but has to rearrange during maturation of the pre-60S ribosomal subunit. Rpf2 and Rrs1 have been implicated in 5S RNP biogenesis, but their precise role was unclear. Here, we present the crystal structure of the Rpf2–Rrs1 complex from Aspergillus nidulans at 1.5 Å resolution and describe it as Brix domain of Rpf2 completed by Rrs1 to form two anticodon-binding domains with functionally important tails. Fitting the X-ray structure into the cryo-EM density of a previously described pre-60S particle correlates with biochemical data. The heterodimer forms specific contacts with the 5S rRNA, RpL5 and the biogenesis factor Rsa4. The flexible protein tails of Rpf2–Rrs1 localize to the central protuberance. Two helices in the Rrs1 C-terminal tail occupy a strategic position to block the rotation of 25S rRNA and the 5S RNP. Our data provide a structural model for 5S RNP recruitment to the pre-60S particle and explain why removal of Rpf2–Rrs1 is necessary for rearrangements to drive 60S maturation. PMID:26117542

  4. Repositioning of antibiotic levofloxacin as a mitochondrial biogenesis inhibitor to target breast cancer.

    PubMed

    Yu, Min; Li, Ruishu; Zhang, Juan

    2016-03-18

    Targeting mitochondrial biogenesis has become a potential therapeutic strategy in cancer due to their unique metabolic dependencies. In this study, we show that levofloxacin, a FDA-approved antibiotic, is an attractive candidate for breast cancer treatment. This is achieved by the inhibition of proliferation and induction of apoptosis in a panel of breast cancer cell lines while sparing normal breast cells. It also acts synergistically with conventional chemo drug in two independent in vivo breast xenograft mouse models. Importantly, levofloxacin inhibits mitochondrial biogenesis as shown by the decreased level of mitochondrial respiration, membrane potential and ATP. In addition, the anti-proliferative and pro-apoptotic effects of levofloxacin are reversed by acetyl-L-Carnitine (ALCAR, a mitochondrial fuel), confirming that levofloxacin's action in breast cancer cells is through inhibition of mitochondrial biogenesis. A consequence of mitochondrial biogenesis inhibition by levofloxacin in breast cancer cells is the deactivation of PI3K/Akt/mTOR and MAPK/ERK pathways. We further demonstrate that breast cancer cells have increased mitochondrial biogenesis than normal breast cells, and this explains their different sensitivity to levofloxacin. Our work suggest that levofloxacin is a useful addition to breast cancer treatment. Our work also establish the essential role of mitochondrial biogenesis on the activation of PI3K/Akt/mTOR and MAPK/ERK pathways in breast cancer cells. PMID:26902121

  5. Mitochondrial cytochrome c biogenesis: no longer an enigma

    PubMed Central

    Babbitt, Shalon E.; Sutherland, Molly C.; Francisco, Brian San; Mendez, Deanna L.; Kranz, Robert G.

    2015-01-01

    Cytochromes c and c1are heme proteins that are essential for aerobic respiration. Release of cytochrome c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for import of apocytochrome c. Thus, HCCS affects cellular levels of cytochrome c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles played by heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways. PMID:26073510

  6. The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit.

    PubMed

    Gudavicius, Geoff; Dilworth, David; Serpa, Jason J; Sessler, Nicole; Petrotchenko, Evgeniy V; Borchers, Christoph H; Nelson, Christopher J

    2014-07-01

    Peptidyl-proline isomerases of the FK506-binding protein (FKBP) family belong to a class of enzymes that catalyze the cis-trans isomerization of prolyl-peptide bonds in proteins. A handful of FKBPs are found in the nucleus, implying that the isomerization of proline in nuclear proteins is enzymatically controlled. FKBP25 is a nuclear protein that has been shown to associate with chromatin modifiers and transcription factors. In this study, we performed the first proteomic characterization of FKBP25 and found that it interacts with numerous ribosomal proteins, ribosomal processing factors, and a small selection of chromatin modifiers. In agreement with previous reports, we found that nucleolin is a major FKBP25-interacting protein and demonstrated that this interaction is dependent on rRNA. FKBP25 interacts with the immature large ribosomal subunit in nuclear extract but does not associate with mature ribosomes, implicating this FKBP's action in ribosome biogenesis. Despite engaging nascent 60S ribosomes, FKBP25 does not affect steady-state levels of rRNAs or its pre-rRNA intermediates. We conclude that FKBP25 is likely recruited to preribosomes to chaperone one of the protein components of the ribosome large subunit. PMID:24840943

  7. Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing

    PubMed Central

    Hang, Runlai; Liu, Chunyan; Ahmad, Ayaz; Zhang, Yong; Lu, Falong; Cao, Xiaofeng

    2014-01-01

    Ribosome biogenesis is a fundamental and tightly regulated cellular process, including synthesis, processing, and assembly of rRNAs with ribosomal proteins. Protein arginine methyltransferases (PRMTs) have been implicated in many important biological processes, such as ribosome biogenesis. Two alternative precursor rRNA (pre-rRNA) processing pathways coexist in yeast and mammals; however, how PRMT affects ribosome biogenesis remains largely unknown. Here we show that Arabidopsis PRMT3 (AtPRMT3) is required for ribosome biogenesis by affecting pre-rRNA processing. Disruption of AtPRMT3 results in pleiotropic developmental defects, imbalanced polyribosome profiles, and aberrant pre-rRNA processing. We further identify an alternative pre-rRNA processing pathway in Arabidopsis and demonstrate that AtPRMT3 is required for the balance of these two pathways to promote normal growth and development. Our work uncovers a previously unidentified function of PRMT in posttranscriptional regulation of rRNA, revealing an extra layer of complexity in the regulation of ribosome biogenesis. PMID:25352672

  8. EVALUATION OF THE BIOGENESIS SOIL WASHING TECHNOLOGY

    EPA Science Inventory

    The BioGenesis Enterprises, Inc. (BioGenesis) soil washing technology was demonstrated as part of the US Environmental Protection Agency's (EPA) Superfund Innovative Technology Evaluation (SITE) program in November 1992. The demonstration was conducted over three days at a petrol...

  9. MicroRNA-27b Regulates Mitochondria Biogenesis in Myocytes

    PubMed Central

    Zhang, Shunhua; Du, Jingjing; Bai, Lin; Zhang, Yi; Jiang, Yanzhi; Li, Xuewei; Wang, Jinyong; Zhu, Li

    2016-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that affect the post-transcriptional regulation of various biological pathways. To date, it is not fully understood how miRNAs regulate mitochondrial biogenesis. This study aimed at the identification of the role of miRNA-27b in mitochondria biogenesis. The mitochondria content in C2C12 cells was significantly increased during myogenic differentiation and accompanied by a marked decrease of miRNA-27b expression. Furthermore, the expression of the predicted target gene of miRNA-27b, forkhead box j3 (Foxj3), was also increased during myogenic differentiation. Luciferase activity assays confirmed that miRNA-27b directly targets the 3’-untranslated region (3’-UTR) of Foxj3. Overexpression of miRNA-27b provoked a decrease of mitochondria content and diminished expression of related mitochondrial genes and Foxj3 both at mRNA and protein levels. The expression levels of downstream genes of Foxj3, such as Mef2c, PGC1α, NRF1 and mtTFA, were also decreased in C2C12 cells upon overexpression of miRNA-27b. These results suggested that miRNA-27b may affect mitochondria biogenesis by down-regulation of Foxj3 during myocyte differentiation. PMID:26849429

  10. Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis.

    PubMed

    Talkish, Jason; Biedka, Stephanie; Jakovljevic, Jelena; Zhang, Jingyu; Tang, Lan; Strahler, John R; Andrews, Philip C; Maddock, Janine R; Woolford, John L

    2016-06-01

    In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of nascent pre-rRNAs are processed cotranscriptionally, with cleavage at the A2 site first releasing a pre-40S ribosomal subunit followed by release of a pre-60S ribosomal subunit upon transcription termination. Ribosome assembly is driven in part by hierarchical association of assembly factors and r-proteins. Groups of proteins are thought to associate with pre-ribosomes cotranscriptionally during early assembly steps, whereas others associate later, after transcription is completed. Here we describe a previously uncharacterized phenotype observed upon disruption of ribosome assembly, in which normally late-binding proteins associate earlier, with pre-ribosomes containing 35S pre-rRNA. As previously observed by many other groups, we show that disruption of 60S subunit biogenesis results in increased amounts of 35S pre-rRNA, suggesting that a greater fraction of pre-rRNAs are processed post-transcriptionally. Surprisingly, we found that early pre-ribosomes containing 35S pre-rRNA also contain proteins previously thought to only associate with pre-ribosomes after early pre-rRNA processing steps have separated maturation of the two subunits. We believe the shift to post-transcriptional processing is ultimately due to decreased cellular division upon disruption of ribosome assembly. When cells are grown under stress or to high density, a greater fraction of pre-rRNAs are processed post-transcriptionally and follow an alternative processing pathway. Together, these results affirm the principle that ribosome assembly occurs through different, parallel assembly pathways and suggest that there is a kinetic foot-race between the formation of protein binding sites and pre-rRNA processing events. PMID:27036125

  11. Ribosome biogenesis in the yeast Saccharomyces cerevisiae.

    PubMed

    Woolford, John L; Baserga, Susan J

    2013-11-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

  12. The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

    PubMed Central

    Keller, Michael; Taskin, Asli A.; Horvath, Susanne E.; Guan, Xue Li; Prinz, Claudia; Opalińska, Magdalena; Zorzin, Carina; van der Laan, Martin; Wenk, Markus R.; Schubert, Rolf; Wiedemann, Nils; Holzer, Martin

    2015-01-01

    Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein. PMID:26347140

  13. Biogenesis of the multifunctional lipid droplet: Lipids, proteins, and sites

    PubMed Central

    Gross, Steven P.

    2014-01-01

    Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle’s cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis. PMID:24590170

  14. Outer membrane lipoprotein biogenesis: Lol is not the end.

    PubMed

    Konovalova, Anna; Silhavy, Thomas J

    2015-10-01

    Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology. PMID:26370942

  15. The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

    NASA Technical Reports Server (NTRS)

    Morowitz, Harold; Peterson, Eta; Chang, Sherwood

    1995-01-01

    This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

  16. Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

    PubMed Central

    Babiano, Reyes; Badis, Gwenael; Saveanu, Cosmin; Namane, Abdelkader; Doyen, Antonia; Díaz-Quintana, Antonio; Jacquier, Alain; Fromont-Racine, Micheline; de la Cruz, Jesús

    2013-01-01

    Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation. PMID:23945946

  17. HDL biogenesis, remodeling, and catabolism.

    PubMed

    Zannis, Vassilis I; Fotakis, Panagiotis; Koukos, Georgios; Kardassis, Dimitris; Ehnholm, Christian; Jauhiainen, Matti; Chroni, Angeliki

    2015-01-01

    In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research. PMID:25522986

  18. Selenite Stimulates Mitochondrial Biogenesis Signaling and Enhances Mitochondrial Functional Performance in Murine Hippocampal Neuronal Cells

    PubMed Central

    Idris, Haza; Kumari, Santosh; Li, P. Andy

    2012-01-01

    Supplementation of selenium has been shown to protect cells against free radical mediated cell damage. The objectives of this study are to examine whether supplementation of selenium stimulates mitochondrial biogenesis signaling pathways and whether selenium enhances mitochondrial functional performance. Murine hippocampal neuronal HT22 cells were treated with sodium selenite for 24 hours. Mitochondrial biogenesis markers, mitochondrial respiratory rate and activities of mitochondrial electron transport chain complexes were measured and compared to non-treated cells. The results revealed that treatment of selenium to the HT22 cells elevated the levels of nuclear mitochondrial biogenesis regulators PGC-1α and NRF1, as well as mitochondrial proteins cytochrome c and cytochrome c oxidase IV (COX IV). These effects are associated with phosphorylation of Akt and cAMP response element-binding (CREB). Supplementation of selenium significantly increased mitochondrial respiration and improved the activities of mitochondrial respiratory complexes. We conclude that selenium activates mitochondrial biogenesis signaling pathway and improves mitochondrial function. These effects may be associated with modulation of AKT-CREB pathway. PMID:23110128

  19. Getting ready for building: signaling and autophagosome biogenesis.

    PubMed

    Abada, Adi; Elazar, Zvulun

    2014-08-01

    Autophagy is the main cellular catabolic process responsible for degrading organelles and large protein aggregates. It is initiated by the formation of a unique membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double-membrane vesicle termed the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and degradation of the inner membrane contents complete the process. The extent of autophagy must be tightly regulated to avoid destruction of proteins and organelles essential for cell survival. Autophagic activity is thus regulated by external and internal cues, which initiate the formation of well-defined autophagy-related protein complexes that mediate autophagosome formation and selective cargo recruitment into these organelles. Autophagosome formation and the signaling pathways that regulate it have recently attracted substantial attention. In this review, we analyze the different signaling pathways that regulate autophagy and discuss recent progress in our understanding of autophagosome biogenesis. PMID:25027988

  20. Insights into chloroplast biogenesis and development.

    PubMed

    Pogson, Barry J; Ganguly, Diep; Albrecht-Borth, Verónica

    2015-09-01

    In recent years many advances have been made to obtain insight into chloroplast biogenesis and development. In plants several plastids types exist such as the proplastid (which is the progenitor of all plastids), leucoplasts (group of colourless plastids important for storage including elaioplasts (lipids), amyloplasts (starch) or proteinoplasts (proteins)), chromoplasts (yellow to orange-coloured due to carotenoids, in flowers or in old leaves as gerontoplasts), and the green chloroplasts. Chloroplasts are indispensable for plant development; not only by performing photosynthesis and thus rendering the plant photoautotrophic, but also for biochemical processes (which in some instances can also take place in other plastids types), such as the synthesis of pigments, lipids, and plant hormones and sensing environmental stimuli. Although we understand many aspects of these processes there are gaps in our understanding of the establishment of functional chloroplasts and their regulation. Why is that so? Even though chloroplast function is comparable in all plants and most of the algae, ferns and moss, detailed analyses have revealed many differences, specifically with respect to its biogenesis. As an update to our prior review on the genetic analysis of chloroplast biogenesis and development [1] herein we will focus on recent advances in Angiosperms (monocotyledonous and dicotyledonous plants) that provide novel insights and highlight the challenges and prospects for unravelling the regulation of chloroplast biogenesis specifically during the establishment of the young plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25667967

  1. Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

    PubMed

    Scala, F; Brighenti, E; Govoni, M; Imbrogno, E; Fornari, F; Treré, D; Montanaro, L; Derenzini, M

    2016-02-25

    Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins-MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate. PMID:25961931

  2. Biological applications of hydrophilic C60 derivatives (hC60s)- a structural perspective.

    PubMed

    Zhu, Xiaolei; Sollogoub, Matthieu; Zhang, Yongmin

    2016-06-10

    Reactive oxygen species (ROS) generation and radical scavenging are dual properties of hydrophilic C60 derivatives (hC60s). hC60s eliminate radicals in dark, while they produce reactive oxygen species (ROS) in the presence of irradiation and oxygen. Compared to the pristine C60 suspension, the aqueous solution of hC60s is easier to handle in vivo. hC60s are diverse and could be placed into two general categories: covalently modified C60 derivatives and pristine C60 solubilized non-covalently by macromolecules. In order to present in detail, the above categories are broken down into 8 parts: C60(OH)n, C60 with carboxylic acid, C60 with quaternary ammonium salts, C60 with peptide, C60 containing sugar, C60 modified covalently or non-covalently solubilized by cyclodextrins (CDs), pristine C60 delivered by liposomes, functionalized C60-polymer and pristine C60 solubilized by polymer. Each hC60 shows the propensity to be ROS producer or radical scavenger. This preference is dependent on hC60s structures. For example, major application of C60(OH)n is radical scavenger, while pristine C60/γ-CD complex usually serves as ROS producer. In addition, the electron acceptability and innate hydrophobic surface confer hC60s with O2 uptake inhibition, HIV inhibition and membrane permeability. In this review, we summarize the preparation methods and biological applications of hC60s according to the structures. PMID:27049677

  3. Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.

    PubMed

    Stiller, Sebastian B; Höpker, Jan; Oeljeklaus, Silke; Schütze, Conny; Schrempp, Sandra G; Vent-Schmidt, Jens; Horvath, Susanne E; Frazier, Ann E; Gebert, Natalia; van der Laan, Martin; Bohnert, Maria; Warscheid, Bettina; Pfanner, Nikolaus; Wiedemann, Nils

    2016-05-10

    The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane. PMID:27166948

  4. GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

    PubMed

    Shiromoto, Yusuke; Kuramochi-Miyagawa, Satomi; Daiba, Akito; Chuma, Shinichiro; Katanaya, Ami; Katsumata, Akiko; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakamura, Toshinobu; Yoshinaga, Koichi; Asada, Noriko; Nakamura, Shota; Yasunaga, Teruo; Kojima-Kita, Kanako; Itou, Daisuke; Kimura, Tohru; Nakano, Toru

    2013-06-01

    piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis. PMID:23611983

  5. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    SciTech Connect

    Zuo, Luning; Li, Qiang; Sun, Bei; Xu, Zhiying; Ge, Zhiming

    2013-03-29

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  6. Vulnerability of microRNA biogenesis in FTD-ALS.

    PubMed

    Eitan, Chen; Hornstein, Eran

    2016-09-15

    The genetics of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) turn our attention to RNA metabolism, primarily because many of the identified diseases-associated genes encode for RNA-binding proteins. microRNAs (miRNAs) are endogenous noncoding RNAs that play critical roles in maintaining brain integrity. The current review sheds light on miRNA dysregulation in neurodegenerative diseases, focusing on FTD-ALS. We propose that miRNAs are susceptible to fail when protein factors that are critical for miRNA biogenesis malfunction. Accordingly, potential insufficiencies of the 'microprocessor' complex, the nucleo-cytoplasmic export of miRNA precursors or their processing by Dicer were recently reported. Furthermore, specific miRNAs are involved in the regulation of pathways that are essential for neuronal survival or function. Any change in the expression of these specific miRNAs or in their ability to recognize their target sequences will have negative consequences. Taken together, recent reports strengthens the hypothesis that dysregulation of miRNAs might play an important role in the pathogenesis of neurodegenerative diseases, and highlights the miRNA biogenesis machinery as an interesting target for therapeutic interventions for ALS as well as FTD. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease. PMID:26778173

  7. Phosphatidylinositol 3-Monophosphate Is Involved in Toxoplasma Apicoplast Biogenesis

    PubMed Central

    Tawk, Lina; Dubremetz, Jean-François; Montcourrier, Philippe; Chicanne, Gaëtan; Merezegue, Fabrice; Richard, Véronique; Payrastre, Bernard; Meissner, Markus; Vial, Henri J.; Roy, Christian

    2011-01-01

    Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs. PMID:21379336

  8. Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

    PubMed

    Tawk, Lina; Dubremetz, Jean-François; Montcourrier, Philippe; Chicanne, Gaëtan; Merezegue, Fabrice; Richard, Véronique; Payrastre, Bernard; Meissner, Markus; Vial, Henri J; Roy, Christian; Wengelnik, Kai; Lebrun, Maryse

    2011-02-01

    Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs. PMID:21379336

  9. Carbon fullerenes (C60s) can induce inflammatory responses in the lung of mice

    SciTech Connect

    Park, Eun-Jung; Kim, Hero; Kim, Younghun; Yi, Jongheop; Choi, Kyunghee; Park, Kwangsik

    2010-04-15

    Fullerenes (C60s) occur in the environment due to natural and anthropogenic sources such as volcanic eruptions, forest fires, and the combustion of carbon-based materials. Recently, production and application of engineered C60s have also rapidly increased in diverse industrial fields and biomedicine due to C60' unique physico-chemical properties, so toxicity assessment on environmental and human health is being evaluated as a valuable work. However, data related to the toxicity of C60s have not been abundant up to now. In this study, we studied the immunotoxic mechanism and change of gene expression caused by the instillation of C60s. As a result, C60s induced an increase in sub G1 and G1 arrest in BAL cells, an increase in pro-inflammatory cytokines such as IL-1, TNF-alpha, and IL-6, and an increase of Th1 cytokines such as IL-12 and IFN-r in BAL fluid. In addition, IgE reached the maximum at 1 day after treatment in both BAL fluid and the blood, and decreased in a time-dependent manner. Gene expression of the MHC class II (H2-Eb1) molecule was stronger than that of the MHC class I (H2-T23), and an increase in T cell distribution was also observed during the experiment period. Furthermore, cell infiltration and expression of tissue damage related genes in lung tissue were constantly observed during the experiment period. Based on this, C60s may induce inflammatory responses in the lung of mice.

  10. Impaired Mitochondrial Biogenesis in Adipose Tissue in Acquired Obesity.

    PubMed

    Heinonen, Sini; Buzkova, Jana; Muniandy, Maheswary; Kaksonen, Risto; Ollikainen, Miina; Ismail, Khadeeja; Hakkarainen, Antti; Lundbom, Jesse; Lundbom, Nina; Vuolteenaho, Katriina; Moilanen, Eeva; Kaprio, Jaakko; Rissanen, Aila; Suomalainen, Anu; Pietiläinen, Kirsi H

    2015-09-01

    Low mitochondrial number and activity have been suggested as underlying factors in obesity, type 2 diabetes, and metabolic syndrome. However, the stage at which mitochondrial dysfunction manifests in adipose tissue after the onset of obesity remains unknown. Here we examined subcutaneous adipose tissue (SAT) samples from healthy monozygotic twin pairs, 22.8-36.2 years of age, who were discordant (ΔBMI >3 kg/m(2), mean length of discordance 6.3 ± 0.3 years, n = 26) and concordant (ΔBMI <3 kg/m(2), n = 14) for body weight, and assessed their detailed mitochondrial metabolic characteristics: mitochondrial-related transcriptomes with dysregulated pathways, mitochondrial DNA (mtDNA) amount, mtDNA-encoded transcripts, and mitochondrial oxidative phosphorylation (OXPHOS) protein levels. We report global expressional downregulation of mitochondrial oxidative pathways with concomitant downregulation of mtDNA amount, mtDNA-dependent translation system, and protein levels of the OXPHOS machinery in the obese compared with the lean co-twins. Pathway analysis indicated downshifting of fatty acid oxidation, ketone body production and breakdown, and the tricarboxylic acid cycle, which inversely correlated with adiposity, insulin resistance, and inflammatory cytokines. Our results suggest that mitochondrial biogenesis, oxidative metabolic pathways, and OXPHOS proteins in SAT are downregulated in acquired obesity, and are associated with metabolic disturbances already at the preclinical stage. PMID:25972572

  11. Cell wall structure and biogenesis in Aspergillus species.

    PubMed

    Yoshimi, Akira; Miyazawa, Ken; Abe, Keietsu

    2016-09-01

    Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections. PMID:27140698

  12. DExD/H-box RNA helicases in ribosome biogenesis

    PubMed Central

    Martin, Roman; Straub, Annika U.; Doebele, Carmen; Bohnsack, Markus T.

    2013-01-01

    Ribosome synthesis requires a multitude of cofactors, among them DExD/H-box RNA helicases. Bacterial RNA helicases involved in ribosome assembly are not essential, while eukaryotes strictly require multiple DExD/H-box proteins that are involved in the much more complex ribosome biogenesis pathway. Here, RNA helicases are thought to act in structural remodeling of the RNPs including the modulation of protein binding, and they are required for allowing access or the release of specific snoRNPs from pre-ribosomes. Interestingly, helicase action is modulated by specific cofactors that can regulate recruitment and enzymatic activity. This review summarizes the current knowledge and focuses on recent findings and open questions on RNA helicase function and regulation in ribosome synthesis. PMID:22922795

  13. TIP47 functions in the biogenesis of lipid droplets

    PubMed Central

    Bulankina, Anna V.; Deggerich, Anke; Wenzel, Dirk; Mutenda, Kudzai; Wittmann, Julia G.; Rudolph, Markus G.; Burger, Koert N.J.

    2009-01-01

    TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs. PMID:19451273

  14. Flexibility in targeting and insertion during bacterial membrane protein biogenesis

    SciTech Connect

    Bloois, Edwin van; Hagen-Jongman, Corinne M. ten; Luirink, Joen

    2007-10-26

    The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.

  15. Mechanism of eIF6 release from the nascent 60S ribosomal subunit.

    PubMed

    Weis, Félix; Giudice, Emmanuel; Churcher, Mark; Jin, Li; Hilcenko, Christine; Wong, Chi C; Traynor, David; Kay, Robert R; Warren, Alan J

    2015-11-01

    SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias. PMID:26479198

  16. Mechanism of eIF6 release from the nascent 60S ribosomal subunit

    PubMed Central

    Weis, Félix; Giudice, Emmanuel; Churcher, Mark; Jin, Li; Hilcenko, Christine; Wong, Chi C; Traynor, David; Kay, Robert R; Warren, Alan J

    2016-01-01

    SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS–EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias. PMID:26479198

  17. Nucleolin protein interacts with microprocessor complex to affect biogenesis of microRNAs 15a and 16.

    PubMed

    Pickering, Brian F; Yu, Dihua; Van Dyke, Michael W

    2011-12-23

    MicroRNAs (miRNA) are endogenous, short, non-coding RNA that undergo a multistep biogenesis before generating the functional, mature sequence. The core components of the microprocessor complex, consisting of Drosha and DGCR8, are both necessary and sufficient for this process, although accessory proteins have been found that modulate the biogenesis of a subset of miRNA. Curiously, many of the proteins involved in miRNA biogenesis are also needed for ribosomal RNA processing. Here we show that nucleolin, another protein critical for rRNA processing, is involved in the biogenesis of microRNA 15a/16 (miR-15a/16), specifically at the primary to precursor stage of processing. Through overexpression and knockdown studies, we show that miR-15a/16 levels are directly correlated to nucleolin expression. Furthermore, we found that cellular localization is critical for the proper functioning of nucleolin in this pathway and that nucleolin directly interacts with DGCR8 and Drosha in the nucleus. Nucleolin can bind to the primary miRNA both directly and specifically. Finally, we show that in the absence of nucleolin, cell extracts are unable to process miR-15a/16 in vitro and that this can be rescued by the addition of nucleolin. Our findings offer a new protein component in the microRNA biogenesis pathway and lend insight into miRNA dysregulation in certain cancers. PMID:22049078

  18. Canonical and alternate functions of the microRNA biogenesis machinery

    PubMed Central

    Chong, Mark M.W.; Zhang, Guoan; Cheloufi, Sihem; Neubert, Thomas A.; Hannon, Gregory J.; Littman, Dan R.

    2010-01-01

    The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem–loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency. PMID:20713509

  19. Canonical and alternate functions of the microRNA biogenesis machinery.

    PubMed

    Chong, Mark M W; Zhang, Guoan; Cheloufi, Sihem; Neubert, Thomas A; Hannon, Gregory J; Littman, Dan R

    2010-09-01

    The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency. PMID:20713509

  20. Utilizing small nutrient compounds as enhancers of exercise-induced mitochondrial biogenesis

    PubMed Central

    Craig, Daniel M.; Ashcroft, Stephen P.; Belew, Micah Y.; Stocks, Ben; Currell, Kevin; Baar, Keith; Philp, Andrew

    2015-01-01

    Endurance exercise, when performed regularly as part of a training program, leads to increases in whole-body and skeletal muscle-specific oxidative capacity. At the cellular level, this adaptive response is manifested by an increased number of oxidative fibers (Type I and IIA myosin heavy chain), an increase in capillarity and an increase in mitochondrial biogenesis. The increase in mitochondrial biogenesis (increased volume and functional capacity) is fundamentally important as it leads to greater rates of oxidative phosphorylation and an improved capacity to utilize fatty acids during sub-maximal exercise. Given the importance of mitochondrial biogenesis for skeletal muscle performance, considerable attention has been given to understanding the molecular cues stimulated by endurance exercise that culminate in this adaptive response. In turn, this research has led to the identification of pharmaceutical compounds and small nutritional bioactive ingredients that appear able to amplify exercise-responsive signaling pathways in skeletal muscle. The aim of this review is to discuss these purported exercise mimetics and bioactive ingredients in the context of mitochondrial biogenesis in skeletal muscle. We will examine proposed modes of action, discuss evidence of application in skeletal muscle in vivo and finally comment on the feasibility of such approaches to support endurance-training applications in humans. PMID:26578969

  1. Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis

    PubMed Central

    Aitchison, Adam J.; Arsenault, Daniel J.; Ridgway, Neale D.

    2015-01-01

    The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs. PMID:26108622

  2. Biogenesis of the cytochrome bc(1) complex and role of assembly factors.

    PubMed

    Smith, Pamela M; Fox, Jennifer L; Winge, Dennis R

    2012-02-01

    The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. PMID:22138626

  3. Reprint of: Biogenesis of the cytochrome bc(1) complex and role of assembly factors.

    PubMed

    Smith, Pamela M; Fox, Jennifer L; Winge, Dennis R

    2012-06-01

    The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. PMID:22564912

  4. Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions

    PubMed Central

    Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

    2016-01-01

    Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis. PMID:26902640

  5. shutdown is a component of the Drosophila piRNA biogenesis machinery

    PubMed Central

    Preall, Jonathan B.; Czech, Benjamin; Guzzardo, Paloma M.; Muerdter, Felix; Hannon, Gregory J.

    2012-01-01

    In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown. PMID:22753781

  6. Centriole biogenesis and function in multiciliated cells

    PubMed Central

    Zhang, Siwei; Mitchell, Brian J.

    2016-01-01

    The use of Xenopus embryonic skin as a model system for the development of ciliated epithelia is well established. This tissue is comprised of numerous cell types, most notably the multiciliated cells (MCCs) that each contain approximately 150 motile cilia. At the base of each cilium lies the centriole-based structure called the basal body. Centriole biogenesis is typically restricted to two new centrioles per cell cycle, each templating from an existing “mother” centriole. In contrast, MCCs are post-mitotic cells in which the majority of centrioles arise “de novo” without templating from a mother centriole, instead, these centrioles nucleate from an electron-dense structure termed the deuterostome. How centriole number is regulated in these cells and the mechanism by which the deuterosome templates nascent centrioles is still poorly understood. Here, we describe methods for regulating MCC cell fate as well as for visualizing and manipulating centriole biogenesis. PMID:26175436

  7. Oil body biogenesis during Brassica napus embryogenesis.

    PubMed

    He, Yu-Qing; Wu, Yan

    2009-08-01

    Although the oil body is known to be an important membrane enclosed compartment for oil storage in seeds, we have little understanding about its biogenesis during embryogenesis. In the present study we investigated the oil body emergence and variations in Brassica napus cv. Topas. The results demonstrate that the oil bodies could be detected already at the heart stage, at the same time as the embryos began to turn green, and the starch grains accumulated in the chloroplast stroma. In comparison, we have studied the development of oil bodies between Arabidopsis thaliana wild type (Col) and the low-seed-oil mutant wrinkled1-3. We observed that the oil body development in the embryos of Col is similar to that of B. napus cv. Topas, and that the size of the oil bodies was obviously smaller in the embryos of wrinkled1-3. Our results suggest that the oil body biogenesis might be coupled with the embryo chloroplast. PMID:19686376

  8. Analysis of photosystem II biogenesis in cyanobacteria.

    PubMed

    Heinz, Steffen; Liauw, Pasqual; Nickelsen, Jörg; Nowaczyk, Marc

    2016-03-01

    Photosystem II (PSII), a large multisubunit membrane protein complex found in the thylakoid membranes of cyanobacteria, algae and plants, catalyzes light-driven oxygen evolution from water and reduction of plastoquinone. Biogenesis of PSII requires coordinated assembly of at least 20 protein subunits, as well as incorporation of various organic and inorganic cofactors. The stepwise assembly process is facilitated by numerous protein factors that have been identified in recent years. Further analysis of this process requires the development or refinement of specific methods for the identification of novel assembly factors and, in particular, elucidation of the unique role of each. Here we summarize current knowledge of PSII biogenesis in cyanobacteria, focusing primarily on the impact of methodological advances and innovations. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux. PMID:26592144

  9. PPARα in lysosomal biogenesis: A perspective

    PubMed Central

    Ghosh, Arunava; Pahan, Kalipada

    2016-01-01

    Lysosomes are membrane-bound vesicles containing hydrolytic enzymes, ubiquitously present in all eukaryotic cells. Classically considered to be central to the cellular waste management machinery, recent studies revealed the role of lysosomes in a wide array of cellular processes like, degradation, cellular development, programmed cell death, secretion, plasma membrane repair, nutritional responses, and lipid metabolism. We recently studied the regulation of TFEB, considered to be the master regulator of lysosomal biogenesis, by activation of peroxisomal proliferator activated receptor α (PPARα), one of the key regulators of lipid metabolism. In this article, we discuss how the recent finding could be put in to perspective with the previous findings that relate lysosomal biogenesis to lipid metabolism, and comment on the possibility of a bi-directional interplay between these two distinct cellular processes upon activation of PPARα. PMID:26621249

  10. The yeast NOP4 gene product is an essential nucleolar protein required for pre-rRNA processing and accumulation of 60S ribosomal subunits.

    PubMed Central

    Sun, C; Woolford, J L

    1994-01-01

    The Saccharomyces cerevisiae NOP4 gene was isolated by screening a lambda gt11 yeast genomic DNA library with a monoclonal antibody against a yeast nucleolar protein. NOP4 encodes a 78 kDa protein that contains two prototypical RNA recognition motifs (RRMs) flanking an imperfect RRM lacking characteristic RNP1 and RNP2 motifs. In addition, there is a fourth incomplete RRM. NOP4 is a single copy essential gene present on chromosome XVI, between RAD1 and PEP4. To examine the function of Nop4p, we constructed a conditional null allele of NOP4 by placing this gene under the control of the glucose-repressible GAL1 promoter. When cells are shifted from galactose-containing medium to glucose-containing medium, NOP4 transcription is terminated, Nop4 protein is depleted and cell growth is impaired. Nop4 protein depletion results in diminished accumulation of 60S ribosomal subunits, assignable to a defect in ribosome biogenesis arising from a lack of production of mature 25S rRNA from 27S precursor rRNA. Images PMID:8039505

  11. Enantiomeric Natural Products: Occurrence and Biogenesis**

    PubMed Central

    Finefield, Jennifer M.; Sherman, David H.; Kreitman, Martin; Williams, Robert M.

    2012-01-01

    In Nature, chiral natural products are usually produced in optically pure form; however, on occasion Nature is known to produce enantiomerically opposite metabolites. These enantiomeric natural products can arise in Nature from a single species, or from different genera and/or species. Extensive research has been carried out over the years in an attempt to understand the biogenesis of naturally occurring enantiomers, however, many fascinating puzzles and stereochemical anomalies still remain. PMID:22555867

  12. Myristoylated CIL-7 regulates ciliary extracellular vesicle biogenesis.

    PubMed

    Maguire, Julie E; Silva, Malan; Nguyen, Ken C Q; Hellen, Elizabeth; Kern, Andrew D; Hall, David H; Barr, Maureen M

    2015-08-01

    The cilium both releases and binds to extracellular vesicles (EVs). EVs may be used by cells as a form of intercellular communication and mediate a broad range of physiological and pathological processes. The mammalian polycystins (PCs) localize to cilia, as well as to urinary EVs released from renal epithelial cells. PC ciliary trafficking defects may be an underlying cause of autosomal dominant polycystic kidney disease (PKD), and ciliary-EV interactions have been proposed to play a central role in the biology of PKD. In Caenorhabditis elegans and mammals, PC1 and PC2 act in the same genetic pathway, act in a sensory capacity, localize to cilia, and are contained in secreted EVs, suggesting ancient conservation. However, the relationship between cilia and EVs and the mechanisms generating PC-containing EVs remain an enigma. In a forward genetic screen for regulators of C. elegans PKD-2 ciliary localization, we identified CIL-7, a myristoylated protein that regulates EV biogenesis. Loss of CIL-7 results in male mating behavioral defects, excessive accumulation of EVs in the lumen of the cephalic sensory organ, and failure to release PKD-2::GFP-containing EVs to the environment. Fatty acylation, such as myristoylation and palmitoylation, targets proteins to cilia and flagella. The CIL-7 myristoylation motif is essential for CIL-7 function and for targeting CIL-7 to EVs. C. elegans is a powerful model with which to study ciliary EV biogenesis in vivo and identify cis-targeting motifs such as myristoylation that are necessary for EV-cargo association and function. PMID:26041936

  13. Peroxisome biogenesis in mammalian cells: The impact of genes and environment.

    PubMed

    Farr, Rebecca L; Lismont, Celien; Terlecky, Stanley R; Fransen, Marc

    2016-05-01

    The initiation and progression of many human diseases are mediated by a complex interplay of genetic, epigenetic, and environmental factors. As all diseases begin with an imbalance at the cellular level, it is essential to understand how various types of molecular aberrations, metabolic changes, and environmental stressors function as switching points in essential communication networks. In recent years, peroxisomes have emerged as important intracellular hubs for redox-, lipid-, inflammatory-, and nucleic acid-mediated signaling pathways. In this review, we focus on how nature and nurture modulate peroxisome biogenesis and function in mammalian cells. First, we review emerging evidence that changes in peroxisome activity can be linked to the epigenetic regulation of cell function. Next, we outline how defects in peroxisome biogenesis may directly impact cellular pathways involved in the development of disease. In addition, we discuss how changes in the cellular microenvironment can modulate peroxisome biogenesis and function. Finally, given the importance of peroxisome function in multiple aspects of health, disease, and aging, we highlight the need for more research in this still understudied field. PMID:26305119

  14. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

    PubMed

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K; Osvath, Sarah R; Cárcamo-Oyarce, Gerardo; Gloag, Erin S; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G; Cavaliere, Rosalia; Ahrens, Christian H; Charles, Ian G; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  15. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

    PubMed Central

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L.; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K.; Osvath, Sarah R.; Cárcamo-Oyarce, Gerardo; Gloag, Erin S.; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G.; Cavaliere, Rosalia; Ahrens, Christian H.; Charles, Ian G.; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B.

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  16. Organelle biogenesis and interorganellar connections: Better in contact than in isolation.

    PubMed

    Daniele, Tiziana; Schiaffino, Maria Vittoria

    2014-01-01

    Membrane contact sites (MCSs) allow the exchange of molecules and information between organelles, even when their membranes cannot fuse directly. In recent years, a number of functions have been attributed to these contacts, highlighting their critical role in cell homeostasis. Although inter-organellar connections typically involve the endoplasmic reticulum (ER), we recently reported the presence of a novel MCSs between melanosomes and mitochondria. Melanosome-mitochondrion contacts appear mediated by fibrillar bridges resembling the protein tethers linking mitochondria and the ER, both for their ultrastructural features and the involvement of Mitofusin 2. The frequency of these connections correlates spatially and timely with melanosome biogenesis, suggesting a functional link between the 2 processes and in general that organelle biogenesis in the secretory pathway requires interorganellar crosstalks at multiple steps. Here, we summarize the different functions attributed to MCSs, and discuss their possible relevance for the newly identified melanosome-mitochondrion liaison. PMID:25346798

  17. Stomatin-Like Protein 2 Binds Cardiolipin and Regulates Mitochondrial Biogenesis and Function▿

    PubMed Central

    Christie, Darah A.; Lemke, Caitlin D.; Elias, Isaac M.; Chau, Luan A.; Kirchhof, Mark G.; Li, Bo; Ball, Eric H.; Dunn, Stanley D.; Hatch, Grant M.; Madrenas, Joaquín

    2011-01-01

    Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function. PMID:21746876

  18. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

    PubMed Central

    Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

    2007-01-01

    Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

  19. RNase III-independent microRNA biogenesis in mammalian cells

    PubMed Central

    Maurin, Thomas; Cazalla, Demián; Yang, Jr-Shiuan; Bortolamiol-Becet, Diane; Lai, Eric C.

    2012-01-01

    RNase III enzymes are fundamental to the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in all species studied. Although alternative miRNA pathways independent of Drosha or Dicer exist, each still requires one RNase III-type enzyme. Here, we describe two strategies that marry either RNase Z or the Integrator complex with the slicing activity of Argonaute2 to generate highly functional mature miRNAs. We provide stringent validation of their RNase III independence by demonstrating efficient miRNA biogenesis and activity in Drosha and Dicer knockout cells. These data provide proof-of-principle evidence for additional mechanistic possibilities for efficient generation of small regulatory RNAs, and represent novel silencing triggers that may be exploited for technical purposes. PMID:23097423

  20. Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis

    PubMed Central

    Wang, Minshi; Anikin, Leonid; Pestov, Dimitri G.

    2014-01-01

    Ribosome biogenesis is a dynamic multistep process, many features of which are still incompletely documented. Here, we show that changes in this pathway can be captured and annotated by means of a graphic set of pre-rRNA ratios, a technique we call Ratio Analysis of Multiple Precursors (RAMP). We find that knocking down a ribosome synthesis factor produces a characteristic RAMP profile that exhibits consistency across a range of depletion levels. This facilitates the inference of affected steps and simplifies comparative analysis. We applied RAMP to examine how endonucleolytic cleavages of the mouse pre-rRNA transcript in the internal transcribed spacer 1 (ITS1) are affected by depletion of factors required for maturation of the small ribosomal subunit (Rcl1, Fcf1/Utp24, Utp23) and the large subunit (Pes1, Nog1). The data suggest that completion of early maturation in a subunit triggers its release from the common pre-rRNA transcript by stimulating cleavage at the proximal site in ITS1. We also find that splitting of pre-rRNA in the 3′ region of ITS1 is prevalent in adult mouse tissues and quiescent cells, as it is in human cells. We propose a model for subunit separation during mammalian ribosome synthesis and discuss its implications for understanding pre-rRNA processing pathways. PMID:25190460

  1. Order within a mosaic distribution of mitochondrial c-type cytochrome biogenesis systems?

    PubMed

    Allen, James W A; Jackson, Andrew P; Rigden, Daniel J; Willis, Antony C; Ferguson, Stuart J; Ginger, Michael L

    2008-05-01

    Mitochondrial cytochromes c and c(1) are present in all eukaryotes that use oxygen as the terminal electron acceptor in the respiratory chain. Maturation of c-type cytochromes requires covalent attachment of the heme cofactor to the protein, and there are at least five distinct biogenesis systems that catalyze this post-translational modification in different organisms and organelles. In this study, we use biochemical data, comparative genomic and structural bioinformatics investigations to provide a holistic view of mitochondrial c-type cytochrome biogenesis and its evolution. There are three pathways for mitochondrial c-type cytochrome maturation, only one of which is present in prokaryotes. We analyze the evolutionary distribution of these biogenesis systems, which include the Ccm system (System I) and the enzyme heme lyase (System III). We conclude that heme lyase evolved once and, in many lineages, replaced the multicomponent Ccm system (present in the proto-mitochondrial endosymbiont), probably as a consequence of lateral gene transfer. We find no evidence of a System III precursor in prokaryotes, and argue that System III is incompatible with multi-heme cytochromes common to bacteria, but absent from eukaryotes. The evolution of the eukaryotic-specific protein heme lyase is strikingly unusual, given that this protein provides a function (thioether bond formation) that is also ubiquitous in prokaryotes. The absence of any known c-type cytochrome biogenesis system from the sequenced genomes of various trypanosome species indicates the presence of a third distinct mitochondrial pathway. Interestingly, this system attaches heme to mitochondrial cytochromes c that contain only one cysteine residue, rather than the usual two, within the heme-binding motif. The isolation of single-cysteine-containing mitochondrial cytochromes c from free-living kinetoplastids, Euglena and the marine flagellate Diplonema papillatum suggests that this unique form of heme attachment

  2. Biogenesis, delivery, and function of extracellular RNA.

    PubMed

    Patton, James G; Franklin, Jeffrey L; Weaver, Alissa M; Vickers, Kasey; Zhang, Bing; Coffey, Robert J; Ansel, K Mark; Blelloch, Robert; Goga, Andrei; Huang, Bo; L'Etoille, Noelle; Raffai, Robert L; Lai, Charles P; Krichevsky, Anna M; Mateescu, Bogdan; Greiner, Vanille J; Hunter, Craig; Voinnet, Olivier; McManus, Michael T

    2015-01-01

    The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA. PMID:26320939

  3. Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

    PubMed Central

    Maio, Nunziata; Rouault, Tracey. A.

    2014-01-01

    Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

  4. When ribosomes go bad: diseases of ribosome biogenesis

    PubMed Central

    Freed, Emily F.; Bleichert, Franziska; Dutca, Laura M.; Baserga, Susan J.

    2010-01-01

    Ribosomes are vital for cell growth and survival. Until recently, it was believed that mutations in ribosomes or ribosome biogenesis factors would be lethal, due to the essential nature of these complexes. However, in the last few decades, a number of diseases of ribosome biogenesis have been discovered. It remains a challenge in the field to elucidate the molecular mechanisms underlying them. PMID:20174677

  5. Maintaining Ancient Organelles: Mitochondrial Biogenesis and Maturation

    PubMed Central

    Vega, Rick B.; Horton, Julie L.; Kelly, Daniel P.

    2015-01-01

    The ultrastructure of the cardiac myocyte is remarkable for the high density of mitochondria tightly packed between sarcomeres. This structural organization is designed to provide energy in the form of ATP to fuel normal pump function of the heart. A complex system comprised of regulatory factors and energy metabolic machinery, encoded by both mitochondrial and nuclear genomes, is required for the coordinate control of cardiac mitochondrial biogenesis, maturation, and high-capacity function. This process involves the action of a transcriptional regulatory network that builds and maintains the mitochondrial genome, and to drive the expression of the energy transduction machinery. This finely tuned system is responsive to developmental and physiological cues as well as changes in fuel substrate availability. Deficiency of components critical for mitochondrial energy production frequently manifests as a cardiomyopathic phenotype, underscoring the requirement to maintain high respiration rates in the heart. Although a precise causative role is not clear, there is increasing evidence that perturbations in this regulatory system occur in the hypertrophied and failing heart. This review summarizes current knowledge and highlights recent advances in our understanding of the transcriptional regulatory factors and signaling networks that serve to regulate mitochondrial biogenesis and function in the mammalian heart. PMID:25999422

  6. Integrator mediates the biogenesis of enhancer RNAs

    PubMed Central

    Lai, Fan; Gardini, Alessandro; Zhang, Anda; Shiekhattar, Ramin

    2015-01-01

    Integrator is a multi-subunit complex stably associated with the C-terminal domain (CTD) of RNA polymerase II (RNAPII) 1. Integrator is endowed with a core catalytic RNA endonuclease activity, which is required for the 3′-end processing of non-polyadenylated RNAPII-dependent uridylate-rich small nuclear RNA genes (UsnRNAs) 1. Here, we examined the requirement of Integrator in the biogenesis of transcripts derived from distal regulatory elements (enhancers) involved in tissue- and temporal-specific regulation of gene expression 2–5. Integrator is recruited to enhancers and super-enhancers in a stimulus-dependent manner. Functional depletion of Integrator subunits diminishes the signal-dependent induction of eRNAs and abrogates the stimulus-induced enhancer-promoter chromatin looping. Global nuclear run-on and RNAPII profiling reveals a role for Integrator in 3′-end cleavage of eRNAs primary transcripts leading to transcriptional termination. In the absence of Integrator, eRNAs remain bound to RNAPII and their primary transcripts accumulates. Importantly, the induction of eRNAs and gene expression responsiveness requires the catalytic activity of Integrator complex. We propose a role for Integrator in biogenesis of eRNAs and enhancer function in metazoans. PMID:26308897

  7. Keeping FIT, storing fat: Lipid droplet biogenesis.

    PubMed

    Choudhary, Vineet; Golden, Andy; Prinz, William A

    2016-01-01

    All eukaryotes store excess lipids in organelles known as lipid droplets (LDs), which play central roles in lipid metabolism. Understanding LD biogenesis and metabolism is critical for understanding the pathophysiology of lipid metabolic disorders like obesity and atherosclerosis. LDs are composed of a core of neutral lipids surrounded by a monolayer of phospholipids that often contains coat proteins. Nascent LDs bud from the endoplasmic reticulum (ER) but the mechanism is not known. In this commentary we discuss our recent finding that a conserved family of proteins called fat storage-inducing transmembrane (FIT) proteins is necessary for LDs budding from the ER. In cells lacking FIT proteins, LDs remain in the ER membrane. C. elegans has a single FIT protein (FITM-2), which we found is essential; almost all homozygous fitm-2 animals die as larvae and those that survive to adulthood give rise to embryos that die as L1 and L2 larvae. Homozygous fitm-2 animals have a number of abnormalities including a significant decrease in intestinal LDs and dramatic defects in muscle development. Understanding how FIT proteins mediate LD biogenesis and what roles they play in lipid metabolism and development are fascinating challenges for the future. PMID:27383728

  8. Chronic Arsenic Exposure-Induced Oxidative Stress is Mediated by Decreased Mitochondrial Biogenesis in Rat Liver.

    PubMed

    Prakash, Chandra; Kumar, Vijay

    2016-09-01

    The present study was executed to study the effect of chronic arsenic exposure on generation of mitochondrial oxidative stress and biogenesis in rat liver. Chronic sodium arsenite treatment (25 ppm for 12 weeks) decreased mitochondrial complexes activity in rat liver. There was a decrease in mitochondrial superoxide dismutase (MnSOD) activity in arsenic-treated rats that might be responsible for increased protein and lipid oxidation as observed in our study. The messenger RNA (mRNA) expression of mitochondrial and nuclear-encoded subunits of complexes I (ND1 and ND2) and IV (COX I and COX IV) was downregulated in arsenic-treated rats only. The protein and mRNA expression of MnSOD was reduced suggesting increased mitochondrial oxidative damage after arsenic treatment. There was activation of Bax and caspase-3 followed by release of cytochrome c from mitochondria suggesting induction of apoptotic pathway under oxidative stress. The entire phenomenon was associated with decrease in mitochondrial biogenesis as evident by decreased protein and mRNA expression of nuclear respiratory factor 1 (NRF-1), nuclear respiratory factor 2 (NRF-2), peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), and mitochondrial transcription factor A (Tfam) in arsenic-treated rat liver. The results of the present study indicate that arsenic-induced mitochondrial oxidative stress is associated with decreased mitochondrial biogenesis in rat liver that may present one of the mechanisms for arsenic-induced hepatotoxicity. PMID:26767369

  9. Emerging roles of mitochondria in the evolution, biogenesis, and function of peroxisomes

    PubMed Central

    Mohanty, Abhishek; McBride, Heidi M.

    2013-01-01

    In the last century peroxisomes were thought to have an endosymbiotic origin. Along with mitochondria and chloroplasts, peroxisomes primarily regulate their numbers through the growth and division of pre-existing organelles, and they house specific machinery for protein import. These features were considered unique to endosymbiotic organelles, prompting the idea that peroxisomes were key cellular elements that helped facilitate the evolution of multicellular organisms. The functional similarities to mitochondria within mammalian systems expanded these ideas, as both organelles scavenge peroxide and reactive oxygen species, both organelles oxidize fatty acids, and at least in higher eukaryotes, the biogenesis of both organelles is controlled by common nuclear transcription factors of the PPAR family. Over the last decade it has been demonstrated that the fission machinery of both organelles is also shared, and that both organelles act as critical signaling platforms for innate immunity and other pathways. Taken together it is clear that the mitochondria and peroxisomes are functionally coupled, regulating cellular metabolism and signaling through a number of common mechanisms. However, recent work has focused primarily on the role of the ER in the biogenesis of peroxisomes, potentially overshadowing the critical importance of the mitochondria as a functional partner. In this review, we explore the mechanisms of functional coupling of the peroxisomes to the mitochondria/ER networks, providing some new perspectives on the potential contribution of the mitochondria to peroxisomal biogenesis. PMID:24133452

  10. Redox and Reactive Oxygen Species Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

    PubMed Central

    Bourens, Myriam; Fontanesi, Flavia; Soto, Iliana C.; Liu, Jingjing

    2013-01-01

    Abstract Significance: Cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is the major oxygen consumer enzyme in the cell. COX biogenesis involves several redox-regulated steps. The process is highly regulated to prevent the formation of pro-oxidant intermediates. Recent Advances: Regulation of COX assembly involves several reactive oxygen species and redox-regulated steps. These include: (i) Intricate redox-controlled machineries coordinate the expression of COX isoenzymes depending on the environmental oxygen concentration. (ii) COX is a heme A-copper metalloenzyme. COX copper metallation involves the copper chaperone Cox17 and several other recently described cysteine-rich proteins, which are oxidatively folded in the mitochondrial intermembrane space. Copper transfer to COX subunits 1 and 2 requires concomitant transfer of redox power. (iii) To avoid the accumulation of reactive assembly intermediates, COX is regulated at the translational level to minimize synthesis of the heme A-containing Cox1 subunit when assembly is impaired. Critical Issues: An increasing number of regulatory pathways converge to facilitate efficient COX assembly, thus preventing oxidative stress. Future Directions: Here we will review on the redox-regulated COX biogenesis steps and will discuss their physiological relevance. Forthcoming insights into the precise regulation of mitochondrial COX biogenesis in normal and stress conditions will likely open future perspectives for understanding mitochondrial redox regulation and prevention of oxidative stress. Antioxid. Redox Signal. 19, 1940–1952. PMID:22937827

  11. AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

    PubMed Central

    Burgess, Jason; Jauregui, Miluska; Tan, Julie; Rollins, Janet; Lallet, Sylvie; Leventis, Peter A.; Boulianne, Gabrielle L.; Chang, Henry C.; Le Borgne, Roland; Krämer, Helmut; Brill, Julie A.

    2011-01-01

     Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules. PMID:21490149

  12. Rab11A Controls the Biogenesis of Birbeck Granules by Regulating Langerin Recycling and Stability

    PubMed Central

    Uzan-Gafsou, Stéphanie; Bausinger, Huguette; Proamer, Fabienne; Monier, Solange; Lipsker, Dan; Cazenave, Jean-Pierre; Goud, Bruno; de la Salle, Henri

    2007-01-01

    The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A–RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system. PMID:17538027

  13. Rab11A controls the biogenesis of Birbeck granules by regulating Langerin recycling and stability.

    PubMed

    Uzan-Gafsou, Stéphanie; Bausinger, Huguette; Proamer, Fabienne; Monier, Solange; Lipsker, Dan; Cazenave, Jean-Pierre; Goud, Bruno; de la Salle, Henri; Hanau, Daniel; Salamero, Jean

    2007-08-01

    The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system. PMID:17538027

  14. Apolipoprotein a1 increases mitochondrial biogenesis through AMP-activated protein kinase.

    PubMed

    Song, Parkyong; Kwon, Yonghoon; Yea, Kyungmoo; Moon, Hyo-Youl; Yoon, Jong Hyuk; Ghim, Jaewang; Hyun, Hyunjung; Kim, Dayea; Koh, Ara; Berggren, Per-Olof; Suh, Pann-Ghill; Ryu, Sung Ho

    2015-09-01

    Apolipoprotein a1, which is a major lipoprotein component of high-density lipoprotein (HDL), was reported to decrease plasma glucose in type 2 diabetes. Although recent studies also have shown that apolipoprotein a1 is involved in triglyceride (TG) metabolism, the mechanisms by which apolipoprotein a1 modulates TG levels remain largely unexplored. Here we demonstrated that apolipoprotein a1 increased mitochondrial DNA and mitochondria contents through sustained AMPK activation in myotubes. This resulted in enhanced fatty acid oxidation and attenuation of free fatty acid-induced insulin resistance features in skeletal muscle. The increment of mitochondria was mediated through induction of transcription factors, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear transcription factor 1 (NRF-1). The inhibition of AMPK by a pharmacological agent inhibited the induction of mitochondrial biogenesis. Increase of AMPK phosphorylation by apolipoprotein a1 occurs through activation of upstream kinase LKB1. Finally, we confirmed that scavenger receptor Class B, type 1 (SR-B1) is an important receptor for apolipoprotein a1 in stimulating AMPK pathway and mitochondrial biogenesis. Our study suggests that apolipoprotein a1 can alleviate obesity related metabolic disease by inducing AMPK dependent mitochondrial biogenesis. PMID:25982508

  15. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site

    PubMed Central

    Calviño, Fabiola R.; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-01-01

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1–RpL5-N–RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1–RpL5–RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP. PMID:25849277

  16. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

    PubMed

    Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-01-01

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP. PMID:25849277

  17. Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

    SciTech Connect

    Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul; Pestka, James J.

    2009-06-01

    Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

  18. Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

    PubMed

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-03-11

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  19. Nuclear/Nucleolar GTPase 2 Proteins as a Subfamily of YlqF/YawG GTPases Function in Pre-60S Ribosomal Subunit Maturation of Mono- and Dicotyledonous Plants*

    PubMed Central

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I. Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-01-01

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with kcat values 0.12 ± 0.007 min−1 (n = 6) and 0.087 ± 0.002 min−1 (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  20. Determinants of pore folding in potassium channel biogenesis

    PubMed Central

    Delaney, Erin; Khanna, Pooja; Tu, LiWei; Robinson, John M.; Deutsch, Carol

    2014-01-01

    Many ion channels, both selective and nonselective, have reentrant pore loops that contribute to the architecture of the permeation pathway. It is a fundamental feature of these diverse channels, regardless of whether they are gated by changes of membrane potential or by neurotransmitters, and is critical to function of the channel. Misfolding of the pore loop leads to loss of trafficking and expression of these channels on the cell surface. Mature tetrameric potassium channels contain an α-helix within the pore loop. We systematically mutated the “pore helix” residues of the channel Kv1.3 and assessed the ability of the monomer to fold into a tertiary reentrant loop. Our results show that pore loop residues form a canonical α-helix in the monomer early in biogenesis and that disruption of tertiary folding is caused by hydrophilic substitutions only along one face of this α-helix. These results provide insight into the determinants of the reentrant pore conformation, which is essential for ion channel function. PMID:24616516

  1. Enhanced mitochondrial biogenesis contributes to Wnt induced osteoblastic differentiation of C3H10T1/2 cells.

    PubMed

    An, Jee Hyun; Yang, Jae-Yeon; Ahn, Byung Yong; Cho, Sun Wook; Jung, Ju Yeon; Cho, Hwa Young; Cho, Young Min; Kim, Sang Wan; Park, Kyong Soo; Kim, Seong Yeon; Lee, Hong Kyu; Shin, Chan Soo

    2010-07-01

    Mitochondria play a key role in cell physiology including cell differentiation and proliferation. We investigated the changes of mitochondrial biogenesis during Wnt-induced osteoblastic differentiation of murine mesenchymal C3H10T1/2 cells. Scanning electron microscopy demonstrated that activation of Wnt signaling by Wnt-3A conditioned medicum (CM) resulted in significant increase in the number of mitochondria in C3H10T1/2 cells. In addition, the induction of alkaline phosphatase (ALP) activities by Wnt-3A CM was accompanied by significant increase in mitochondrial mass (p<0.05), mitochondrial membrane potential (p<0.05), intracellular reactive oxygen species production (p<0.05), resting oxygen consumption rate (p<0.05), cellular ATP content (p< or =0.05) and mtDNA copy number (p<0.05) compared to the cells with control CM (L292-CM) treatment. Moreover, co-treatment with Dkk-1 or WIF-1, both of which are Wnt inhibitors, abrogated the Wnt-3A-induced ALP activities as well as mitochondrial biogenesis markers. Upregulation of mitochondrial biogenesis by overexpression of mitochondrial transcription factor A (Tfam) significantly enhanced Wnt-induced osteogenesis as measured by ALP activities. In contrast, inhibition of mitochondrial biogenesis by treatment with Zidovudine (AZT) resulted in significant inhibition of ALP activities. Finally, ALP activities in human osteosarcoma cell line devoid of mitochondrial DNA (rho(0) cells) was significantly suppressed both in basal and Wnt-3A stimulated state compared to those from mitochondria-intact cells (rho+ cells). As a mechanism for Wnt-mediated mitochondrial biogenesis, we found that Wnt increased the expression of PGC-1alpha, a critical molecules in mitochondrial biogenesis, through Erk and p38 MAPK pathway independent of beta-catenin signaling. We also found that increased mitochondrial biogenesis is in turn positively regulating TOPflash reporter activity as well as beta-catenin levels. To summarize, mitochodrial

  2. Biogenesis of an Antitumor Antibiotic Protein, Neocarzonostatin

    PubMed Central

    Kudo, Kozo; Kikuchi, Mikio; Ishida, Nakao

    1972-01-01

    A study of the biogenesis of the antitumor protein antibiotic neocarzinostatin (NCS) was undertaken. The production of NCS, as well as the growth of Streptomyces carzinostaticus in a production medium, was sensitive to puromycin, chloramphenicol, and actinomycin D. However, when a 12-hr culture in production medium was transferred to a nongrowth medium consisting of a phosphate buffer with Mg2+ and Ca2+, rapid NCS synthesis and liberation occurred. NCS production in this medium was no longer sensitive to actinomycin D, but was sensitive to puromycin and chloramphenicol. The conversion of a precursor NCS to an active form was shown to occur in this medium. Subcellular analysis suggested that NCS synthesis occurred by a mechanism similar to that of protein synthesis by membrane polysomes. PMID:4670693

  3. ABCA1 and nascent HDL biogenesis.

    PubMed

    Wang, Shuhui; Smith, Jonathan D

    2014-01-01

    ABCA1 mediates the secretion of cellular free cholesterol and phospholipids to an extracellular acceptor, apolipoprotein AI, to form nascent high-density lipoprotein (HDL). Thus, ABCA1 is a key molecule in cholesterol homeostasis. Functional studies of certain Tangier disease mutations demonstrate that ABCA1 has multiple activities, including plasma membrane remodeling and apoAI binding to cell surface, which participate in nascent HDL biogenesis. Recent advances in our understanding of ABCA1 have demonstrated that ABCA1also mediates unfolding the N terminus of apoAI on the cell surface, followed by lipidation of apoAI and release of nascent HDL. Although ABCA1-mediated cholesterol efflux to apoAI can occur on the plasma membrane, the role of apoAI retroendocytosis during cholesterol efflux may play a role in macrophage foam cells that store cholesterol esters in cytoplasmic lipid droplets. PMID:25359426

  4. Cytochrome c biogenesis: the Ccm system.

    PubMed

    Sanders, Carsten; Turkarslan, Serdar; Lee, Dong-Woo; Daldal, Fevzi

    2010-06-01

    Cytochromes of c-type contain covalently attached hemes that are formed via thioether bonds between the vinyls of heme b and cysteines within C(1)XXC(2)H motifs of apocytochromes. In diverse organisms this post-translational modification relies on membrane-associated specific biogenesis proteins, referred to as cytochrome c maturation (Ccm) systems. A highly complex version of these systems, Ccm or System I, is found in Gram-negative bacteria, archaea and plant mitochondria. We describe emerging functional interactions between the Ccm components categorized into three conserved modules, and present a mechanistic view of the molecular basis of ubiquitous vinyl-2 approximately Cys(1) and vinyl-4 approximately Cys(2) heme b-apocytochrome thioether bonds in c-type cytochromes. PMID:20382024

  5. Divergent Mitochondrial Biogenesis Responses in Human Cardiomyopathy

    PubMed Central

    Ahuja, Preeti; Wanagat, Jonathan; Wang, Zhihua; Wang, Yibin; Liem, David A.; Ping, Peipei; Antoshechkin, Igor A.; Margulies, Kenneth B.; MacLellan, W. Robb

    2014-01-01

    Background Mitochondria are key players in the development and progression of heart failure (HF). Mitochondrial (mt) dysfunction leads to diminished energy production and increased cell death contributing to the progression of left ventricular (LV) failure. The fundamental mechanisms that underlie mt dysfunction in HF have not been fully elucidated. Methods and Results To characterize mt morphology, biogenesis and genomic integrity in human HF, we investigated LV tissue from non-failing (NF) hearts and end-stage ischemic (ICM) or dilated (DCM) cardiomyopathic hearts. Although mt dysfunction was present in both types of cardiomyopathy, mt were smaller and increased in number in DCM compared to ICM or NF hearts. Mt volume density and mtDNA copy number was increased by ~2-fold (P<0.001) in DCM hearts in comparison to ICM hearts. These changes were accompanied by an increase in the expression of mtDNA-encoded genes in DCM versus no change in ICM. mtDNA repair and antioxidant genes were reduced in failing hearts suggestive of a defective repair and protection system, which may account for the 4.1-fold increase in mtDNA deletion mutations in DCM (P<0.05 vs NF hearts, P<0.05 vs ICM). Conclusions In DCM, mt dysfunction is associated with mtDNA damage and deletions, which could be a consequence of mutating stress coupled with a PGC-1α-dependent stimulus for mt biogenesis. However, this maladaptive compensatory response contributes to additional oxidative damage. Thus, our findings support further investigations into novel mechanisms and therapeutic strategies for mt dysfunction in DCM. PMID:23589024

  6. Arabidopsis NMD3 Is Required for Nuclear Export of 60S Ribosomal Subunits and Affects Secondary Cell Wall Thickening

    PubMed Central

    Chen, Mei-Qin; Zhang, Ai-Hong; Zhang, Quan; Zhang, Bao-Cai; Nan, Jie; Li, Xia; Liu, Na; Qu, Hong; Lu, Cong-Ming; Sudmorgen; Zhou, Yi-Hua; Xu, Zhi-Hong; Bai, Shu-Nong

    2012-01-01

    NMD3 is required for nuclear export of the 60S ribosomal subunit in yeast and vertebrate cells, but no corresponding function of NMD3 has been reported in plants. Here we report that Arabidopsis thaliana NMD3 (AtNMD3) showed a similar function in the nuclear export of the 60S ribosomal subunit. Interference with AtNMD3 function by overexpressing a truncated dominant negative form of the protein lacking the nuclear export signal sequence caused retainment of the 60S ribosomal subunits in the nuclei. More interestingly, the transgenic Arabidopsis with dominant negative interference of AtNMD3 function showed a striking failure of secondary cell wall thickening, consistent with the altered expression of related genes and composition of cell wall components. Observation of a significant decrease of rough endoplasmic reticulum (RER) in the differentiating interfascicular fiber cells of the transgenic plant stems suggested a link between the defective nuclear export of 60S ribosomal subunits and the abnormal formation of the secondary cell wall. These findings not only clarified the evolutionary conservation of NMD3 functions in the nuclear export of 60S ribosomal subunits in yeast, animals and plants, but also revealed a new facet of the regulatory mechanism underlying secondary cell wall thickening in Arabidopsis. This new facet is that the nuclear export of 60S ribosomal subunits and the formation of RER may play regulatory roles in coordinating protein synthesis in cytoplasm and transcription in nuclei. PMID:22558264

  7. Biosynthetic Study on Antihypercholesterolemic Agent Phomoidride: General Biogenesis of Fungal Dimeric Anhydrides.

    PubMed

    Fujii, Ryuya; Matsu, Yusuke; Minami, Atsushi; Nagamine, Shota; Takeuchi, Ichiro; Gomi, Katsuya; Oikawa, Hideaki

    2015-11-20

    To elucidate the general biosynthetic pathway of fungal dimeric anhydrides, a gene cluster for the biosynthesis of the antihy-percholesterolemic agent phomoidride was identified by heterologous expression of candidate genes encoding the highly reducing polyketide synthase, alkylcitrate synthase (ACS), and alkylcitrate dehydratase (ACDH). An in vitro analysis of ACS and ACDH revealed that they give rise to anhydride monomers. Based on the established monomer biosynthesis, we propose a general biogenesis of dimeric anhydrides involving a single donor unit and four acceptor units. PMID:26558485

  8. Hippo signaling regulates Microprocessor and links cell density-dependent miRNA biogenesis to cancer

    PubMed Central

    Mori, Masaki; Triboulet, Robinson; Mohseni, Morvarid; Schlegelmilch, Karin; Shrestha, Kriti; Camargo, Fernando D.; Gregory, Richard I.

    2014-01-01

    SUMMARY Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. PMID:24581491

  9. Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis.

    PubMed

    Boglev, Yeliz; Badrock, Andrew P; Trotter, Andrew J; Du, Qian; Richardson, Elsbeth J; Parslow, Adam C; Markmiller, Sebastian J; Hall, Nathan E; de Jong-Curtain, Tanya A; Ng, Annie Y; Verkade, Heather; Ober, Elke A; Field, Holly A; Shin, Donghun; Shin, Chong H; Hannan, Katherine M; Hannan, Ross D; Pearson, Richard B; Kim, Seok-Hyung; Ess, Kevin C; Lieschke, Graham J; Stainier, Didier Y R; Heath, Joan K

    2013-01-01

    Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death. PMID:23408911

  10. A Synergistic Antiobesity Effect by a Combination of Capsinoids and Cold Temperature Through Promoting Beige Adipocyte Biogenesis.

    PubMed

    Ohyama, Kana; Nogusa, Yoshihito; Shinoda, Kosaku; Suzuki, Katsuya; Bannai, Makoto; Kajimura, Shingo

    2016-05-01

    Beige adipocytes emerge postnatally within the white adipose tissue in response to certain environmental cues, such as chronic cold exposure. Because of its highly recruitable nature and relevance to adult humans, beige adipocytes have gained much attention as an attractive cellular target for antiobesity therapy. However, molecular circuits that preferentially promote beige adipocyte biogenesis remain poorly understood. We report that a combination of mild cold exposure at 17°C and capsinoids, a nonpungent analog of capsaicin, synergistically and preferentially promotes beige adipocyte biogenesis and ameliorates diet-induced obesity. Gain- and loss-of-function studies show that the combination of capsinoids and cold exposure synergistically promotes beige adipocyte development through the β2-adrenoceptor signaling pathway. This synergistic effect on beige adipocyte biogenesis occurs through an increased half-life of PRDM16, a dominant transcriptional regulator of brown/beige adipocyte development. We document a previously unappreciated molecular circuit that controls beige adipocyte biogenesis and suggest a plausible approach to increase whole-body energy expenditure by combining dietary components and environmental cues. PMID:26936964

  11. Architecture of the Rix1-Rea1 checkpoint machinery during pre-60S-ribosome remodeling.

    PubMed

    Barrio-Garcia, Clara; Thoms, Matthias; Flemming, Dirk; Kater, Lukas; Berninghausen, Otto; Baßler, Jochen; Beckmann, Roland; Hurt, Ed

    2016-01-01

    Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes. PMID:26619264

  12. Biogenesis of the Saccharomyces cerevisiae Pheromone a-Factor, from Yeast Mating to Human Disease

    PubMed Central

    Barrowman, Jemima

    2012-01-01

    Summary: The mating pheromone a-factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a-factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a-factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a-factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a-factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a-factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a-factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila. Thus, additional prenylated signaling molecules resembling a-factor, with as-yet-unknown roles in metazoan biology, may await discovery. PMID:22933563

  13. MicroRNA-761 regulates mitochondrial biogenesis in mouse skeletal muscle in response to exercise.

    PubMed

    Xu, Yanli; Zhao, Chaoxian; Sun, Xuewen; Liu, Zhijun; Zhang, Jianzhong

    2015-11-01

    MicroRNAs (miRNAs) have been suggested to play critical roles in skeletal muscle in response to exercise. Previous study has shown that miR-761 was involved in a novel model regulating the mitochondrial network. However, its role in mitochondrial biogenesis remains poorly understood. Therefore, the current study was aimed to examine the effect of miR-761 on mitochondrial biogenesis in skeletal muscle. Real-time quantitative PCR analysis demonstrated that aberrantly expressed miR-761 is involved in exercise activity and miR-761 is decreased by exercise training compared with the sedentary control mice. miR-761 suppresses mitochondrial biogenesis of C2C12 myocytes by targeting the 3'-UTR of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1α). Overexpression of miR-761 was capable of inhibiting the protein expression levels of PGC-1α. Moreover, miR-761 overexpression suppressed the p38 MAPK signaling pathway and down-regulated the expression of phosphorylated MAPK-activated protein kinase-2 (P-MK2), a downstream kinase of p38 MAPK. The phosphorylation of activating transcription factors 2 (ATF2) that plays a functional role in linking the activation of the p38 MAPK pathway to enhanced transcription of the PGC-1α was also inhibited by the overexpression of miR-761. These findings revealed a novel regulation mechanism for miR-761 in skeletal myocytes, and contributed to a better understanding of the modulation of skeletal muscle in response to exercise. PMID:26408907

  14. Diabetes regulates mitochondrial biogenesis and fission in neurons

    PubMed Central

    Edwards, J.L.; Quattrini, A.; Lentz, S.I.; Figueroa-Romero, C.; Cerri, F.; Backus, C.; Hong, Y.; Feldman, E.L.

    2014-01-01

    Aims Normal mitochondrial (Mt) activity is a critical component of neuronal metabolism and function. Disruption of Mt activity by altered Mt fission and fusion is the root cause of both neurodegenerative disorders and Charcot-Marie-Tooth Type 2A inherited neuropathy. The current study addressed the role of Mt fission in the pathogenesis of diabetic neuropathy (DN). Methods Mt biogenesis and fission were assayed in both in vivo and in vitro models of DN. Gene, protein, mitochondrial DNA and ultrastructural analyses were used to assess Mt biogenesis and fission. Results Our data reveal increased Mt biogenesis in dorsal root ganglion (DRG) neurons from diabetic compared to non-diabetic mice. An essential step in Mt biogenesis is Mt fission, regulated by the Mt fission protein Drp1. Evaluation of in vivo diabetic neurons indicated small, fragmented Mt, suggesting increased fission. In vitro studies reveal short-term hyperglycemic exposure increased expression of Drp1. The influence of hyperglycemia-mediated Mt fission on cellular viability was evaluated by knockdown of Drp1. Knockdown of Drp1 resulted in decreased susceptibility to hyperglycemic damage. Conclusions We propose that: 1) Mt undergo biogenesis in response to hyperglycemia, but the increased biogenesis is insufficient to accommodate the metabolic load; 2) hyperglycemia causes an excess of Mt fission, creating small, damaged mitochondria; and 3) reduction of aberrant Mt fission increases neuronal survival and indicates an important role for the fission-fusion equilibrium in the pathogenesis of DN. PMID:19847394

  15. Targeted cancer therapy with ribosome biogenesis inhibitors: a real possibility?

    PubMed Central

    Brighenti, Elisa; Treré, Davide; Derenzini, Massimo

    2015-01-01

    The effects of many chemotherapeutic drugs on ribosome biogenesis have been underestimated for a long time. Indeed, many drugs currently used for cancer treatment – and which are known to either damage DNA or hinder DNA synthesis – have been shown to exert their toxic action mainly by inhibiting rRNA synthesis or maturation. Moreover, there are new drugs that have been proposed recently for cancer chemotherapy, which only hinder ribosome biogenesis without any genotoxic activity. Even though ribosome biogenesis occurs in both normal and cancer cells, whether resting or proliferating, there is evidence that the selective inhibition of ribosome biogenesis may, in some instances, result in a selective damage to neoplastic cells. The higher sensitivity of cancer cells to inhibitors of rRNA synthesis appears to be the consequence of either the loss of the mechanisms controlling the cell cycle progression or the acquisition of activating oncogene and inactivating tumor suppressor gene mutations that up-regulate the ribosome biogenesis rate. This article reviews those cancer cell characteristics on which the selective cancer cell cytotoxicity induced by the inhibitors of ribosome biogenesis is based. PMID:26415219

  16. Repetitive elements regulate circular RNA biogenesis

    PubMed Central

    Wilusz, Jeremy E

    2015-01-01

    It was long assumed that eukaryotic precursor mRNAs (pre-mRNAs) are almost always spliced to generate a linear mRNA that is subsequently translated to produce a protein. However, it is now clear that thousands of protein-coding genes can be non-canonically spliced to produce circular noncoding RNAs, some of which are expressed at much higher levels than their associated linear mRNAs. How then does the splicing machinery decide whether to generate a linear mRNA or a circular RNA? Recent work has revealed that intronic repetitive elements, including sequences derived from transposons, are critical regulators of this decision. In most cases, circular RNA biogenesis appears to be initiated when complementary sequences from 2 different introns base pair to one another. This brings the splice sites from the intervening exon(s) into close proximity and facilitates the backsplicing event that generates the circular RNA. As many pre-mRNAs contain multiple intronic repeats, distinct circular transcripts can be produced depending on which repeats base pair to one another. Intronic repeats are thus critical regulatory sequences that control the functional output of their host genes, and potentially cause the functions of protein-coding genes to be highly divergent across species. PMID:26442181

  17. Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane.

    PubMed

    Chahales, Peter; Hoffman, Paul S; Thanassi, David G

    2016-04-01

    Many bacterial pathogens assemble surface fibers termed pili or fimbriae that facilitate attachment to host cells and colonization of host tissues. The chaperone/usher (CU) pathway is a conserved secretion system that is responsible for the assembly of virulence-associated pili by many different Gram-negative bacteria. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and an integral outer membrane (OM) assembly and secretion platform termed the usher. Nitazoxanide (NTZ), an antiparasitic drug, was previously shown to inhibit the function of aggregative adherence fimbriae and type 1 pili assembled by the CU pathway in enteroaggregativeEscherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenicE. coli(UPEC). UPEC is the primary causative agent of urinary tract infections, and type 1 and P pili mediate colonization of the bladder and kidneys, respectively. By analysis of the different stages of the CU pilus biogenesis pathway, we show that treatment of bacteria with NTZ causes a reduction in the number of usher molecules in the OM, resulting in a loss of pilus assembly on the bacterial surface. In addition, we determine that NTZ specifically prevents proper folding of the usher β-barrel domain in the OM. Our findings demonstrate that NTZ is a pilicide with a novel mechanism of action and activity against diverse CU pathways. This suggests that further development of the NTZ scaffold may lead to new antivirulence agents that target the usher to prevent pilus assembly. PMID:26824945

  18. Roles of Oxidative Stress, Apoptosis, PGC-1α and Mitochondrial Biogenesis in Cerebral Ischemia

    PubMed Central

    Chen, Shang-Der; Yang, Ding-I; Lin, Tsu-Kung; Shaw, Fu-Zen; Liou, Chia-Wei; Chuang, Yao-Chung

    2011-01-01

    The primary physiological function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Overproduction of reactive oxygen species (ROS) as byproducts generated from mitochondria have been implicated in acute brain injuries such as stroke from cerebral ischemia. It was well-documented that mitochondria-dependent apoptotic pathway involves pro- and anti-apoptotic protein binding, release of cytochrome c, leading ultimately to neuronal death. On the other hand, mitochondria also play a role to counteract the detrimental effects elicited by excessive oxidative stress. Recent studies have revealed that oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves peroxisome proliferative activated receptor-γ (PPARγ) co-activator 1α (PGC1-α). PGC1-α is a master regulator of ROS scavenging enzymes including manganese superoxide dismutase 2 and the uncoupling protein 2, both are mitochondrial proteins, and may contribute to neuronal survival. PGC1-α is also involved in mitochondrial biogenesis that is vital for cell survival. Experimental evidence supports the roles of mitochondrial dysfunction and oxidative stress as determinants of neuronal death as well as endogenous protective mechanisms after stroke. This review aims to summarize the current knowledge focusing on the molecular mechanisms underlying cerebral ischemia involving ROS, mitochondrial dysfunction, apoptosis, mitochondrial proteins capable of ROS scavenging, and mitochondrial biogenesis. PMID:22072942

  19. The MITF family of transcription factors: Role in endolysosomal biogenesis, Wnt signaling, and oncogenesis.

    PubMed

    Ploper, Diego; De Robertis, Edward M

    2015-09-01

    Canonical Wnt signaling influences cellular fate and proliferation through inhibition of Glycogen Synthase Kinase (GSK3) and the subsequent stabilization of its many substrates, most notably β-Catenin, a transcriptional co-activator. MITF, a melanoma oncogene member of the microphthalmia family of transcription factors (MiT), was recently found to contain novel GSK3 phosphorylation sites and to be stabilized by Wnt. Other MiT members, TFEB and TFE3, are known to play important roles in cellular clearance pathways by transcriptionally regulating the biogenesis of lysosomes and autophagosomes via activation of CLEAR elements in gene promoters of target genes. Recent studies suggest that MITF can also upregulate many lysosomal genes. MiT family members are dysregulated in cancer and are considered oncogenes, but the underlying oncogenic mechanisms remain unclear. Here we review the role of MiT members, including MITF, in lysosomal biogenesis, and how cancers overexpressing MITF, TFEB or TFE3 could rewire the lysosomal pathway, inhibit cellular senescence, and activate Wnt signaling by increasing sequestration of negative regulators of Wnt signaling in multivesicular bodies (MVBs). Microarray studies suggest that MITF expression inhibits macroautophagy. In melanoma the MITF-driven increase in MVBs generates a positive feedback loop between MITF, Wnt, and MVBs. PMID:26003288

  20. Biogenesis and architecture of arterivirus replication organelles.

    PubMed

    van der Hoeven, Barbara; Oudshoorn, Diede; Koster, Abraham J; Snijder, Eric J; Kikkert, Marjolein; Bárcena, Montserrat

    2016-07-15

    All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double-membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation. PMID

  1. Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases

    PubMed Central

    McCaughan, Urszula M.; Jayachandran, Uma; Shchepachev, Vadim; Chen, Zhuo Angel; Rappsilber, Juri; Tollervey, David; Cook, Atlanta G.

    2016-01-01

    Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 Å. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of Tsr1 into cryo-electron microscopy maps of pre-40S particles shows that a highly acidic surface of Tsr1 is presented on the outside of pre-40S particles, potentially preventing premature binding to 60S subunits. Late pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1. The location of Tsr1 is predicted to block binding by both factors, strongly indicating that removal of Tsr1 is an essential step during cytoplasmic maturation of 40S ribosomal subunits. PMID:27250689

  2. Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases.

    PubMed

    McCaughan, Urszula M; Jayachandran, Uma; Shchepachev, Vadim; Chen, Zhuo Angel; Rappsilber, Juri; Tollervey, David; Cook, Atlanta G

    2016-01-01

    Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 Å. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of Tsr1 into cryo-electron microscopy maps of pre-40S particles shows that a highly acidic surface of Tsr1 is presented on the outside of pre-40S particles, potentially preventing premature binding to 60S subunits. Late pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1. The location of Tsr1 is predicted to block binding by both factors, strongly indicating that removal of Tsr1 is an essential step during cytoplasmic maturation of 40S ribosomal subunits. PMID:27250689

  3. Mitochondrial biogenesis is required for the anchorage-independent survival and propagation of stem-like cancer cells

    PubMed Central

    Peiris-Pagès, Maria; Ozsvari, Bela; Smith, Duncan L.; Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E.; Cappello, Anna Rita; Pezzi, Vincenzo; Lisanti, Michael P.; Sotgia, Federica

    2015-01-01

    Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFβ-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/β-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a ‘safe’ mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse

  4. The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

    PubMed

    Bielczyk-Maczyńska, Ewa; Lam Hung, Laure; Ferreira, Lauren; Fleischmann, Tobias; Weis, Félix; Fernández-Pevida, Antonio; Harvey, Steven A; Wali, Neha; Warren, Alan J; Barroso, Inês; Stemple, Derek L; Cvejic, Ana

    2015-12-01

    Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate. PMID:26624285

  5. The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish

    PubMed Central

    Ferreira, Lauren; Fleischmann, Tobias; Weis, Félix; Fernández-Pevida, Antonio; Harvey, Steven A.; Wali, Neha; Warren, Alan J.; Barroso, Inês; Stemple, Derek L.; Cvejic, Ana

    2015-01-01

    Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9 sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9 sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9 sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9 sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9 sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate. PMID:26624285

  6. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    SciTech Connect

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai; Yang, Ying; Shen, Weili

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

  7. Chemical modulators of ribosome biogenesis as biological probes.

    PubMed

    Stokes, Jonathan M; Brown, Eric D

    2015-12-01

    Small-molecule inhibitors of protein biosynthesis have been instrumental in the dissection of the complexities of ribosome structure and function. Ribosome biogenesis, on the other hand, is a complex and largely enigmatic process for which there is a paucity of chemical probes. Indeed, ribosome biogenesis has been studied almost exclusively using genetic and biochemical approaches without the benefit of small-molecule inhibitors of this process. Here, we provide a perspective on the promise of chemical inhibitors of ribosome assembly for future research. We explore key obstacles that complicate the interpretation of studies aimed at perturbing ribosome biogenesis in vivo using genetic methods, and we argue that chemical inhibitors are especially powerful because they can be used to induce perturbations in a manner that obviates these difficulties. Thus, in combination with leading-edge biochemical and structural methods, chemical probes offer unique advantages toward elucidating the molecular events that define the assembly of ribosomes. PMID:26575239

  8. Role of AAA(+)-proteins in peroxisome biogenesis and function.

    PubMed

    Grimm, Immanuel; Erdmann, Ralf; Girzalsky, Wolfgang

    2016-05-01

    Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function. PMID:26453804

  9. Exercise induces mitochondrial biogenesis after brain ischemia in rats.

    PubMed

    Zhang, Q; Wu, Y; Zhang, P; Sha, H; Jia, J; Hu, Y; Zhu, J

    2012-03-15

    Stroke is a major cause of death worldwide. Previous studies have suggested both exercise and mitochondrial biogenesis contribute to improved post-ischemic recovery of brain function. However, the exact mechanism underlying this effect is unclear. On the other hand, the benefit of exercise-induced mitochondrial biogenesis in brain has been confirmed. In this study, we attempted to determine whether treadmill exercise induces functional improvement through regulation of mitochondrial biogenesis after brain ischemia. We subjected adult male rats to ischemia, followed by either treadmill exercise or non-exercise and analyzed the effect of exercise on the amount of mitochondrial DNA (mtDNA), expression of mitochondrial biogenesis factors, and mitochondrial protein. In the ischemia-exercise group, only peroxisome proliferator activated receptor coactivator-1 (PGC-1) expression was increased significantly after 3 days of treadmill training. However, after 7 days of training, the levels of mtDNA, nuclear respiratory factor 1, NRF-1, mitochondrial transcription factor A, TFAM, and the mitochondrial protein cytochrome C oxidase subunit IV (COXIV) and heat shock protein-60 (HSP60) also increased above levels observed in non-exercised ischemic animals. These changes followed with significant changes in behavioral scores and cerebral infarct volume. The results indicate that exercise can promote mitochondrial biogenesis after ischemic injury, which may serve as a novel component of exercise-induced repair mechanisms of the brain. Understanding the molecular basis for exercise-induced neuroprotection may be beneficial in the development of therapeutic approaches for brain recovery from the ischemic injury. Based upon our findings, stimulation or enhancement of mitochondrial biogenesis may prove a novel neuroprotective strategy in the future. PMID:22266265

  10. Mitochondrial biogenesis and dynamics in the developing and diseased heart

    PubMed Central

    Dorn, Gerald W.; Vega, Rick B.; Kelly, Daniel P.

    2015-01-01

    The mitochondrion is a complex organelle that serves essential roles in energy transduction, ATP production, and a myriad of cellular signaling events. A finely tuned regulatory network orchestrates the biogenesis, maintenance, and turnover of mitochondria. The high-capacity mitochondrial system in the heart is regulated in a dynamic way to generate and consume enormous amounts of ATP in order to support the constant pumping function in the context of changing energy demands. This review describes the regulatory circuitry and downstream events involved in mitochondrial biogenesis and its coordination with mitochondrial dynamics in developing and diseased hearts. PMID:26443844

  11. Rab11a Is Essential for Lamellar Body Biogenesis in the Human Epidermis.

    PubMed

    Reynier, Marie; Allart, Sophie; Gaspard, Elise; Moga, Alain; Goudounèche, Dominique; Serre, Guy; Simon, Michel; Leprince, Corinne

    2016-06-01

    Most of the skin barrier function is attributable to the outermost layer of the epidermis, the stratum corneum, which is composed of flattened, anucleated cells called corneocytes surrounded by a lipid-enriched lamellar matrix. The composition of the stratum corneum is directly dependent on the underlying granular keratinocytes, which are the last living cells in the stratified epidermis. Many components present in the intercorneocyte matrix are delivered by the underlying granular keratinocytes through a secretion process dependent on lysosome-related organelles called lamellar bodies. Because of the importance of lamellar bodies in the maintenance of the epidermal barrier, the mechanisms regulating their biogenesis must be better understood. In this study, we show that the Rab11a GTPase is highly expressed in terminally differentiated keratinocytes, where it is partly associated with lamellar bodies. Rab11a silencing in three-dimensional in vitro reconstructed human epidermis induces a barrier defect, a decrease in the amount of lipid found in the stratum corneum, a reduction in lamellar body density and secretion areas in granular keratinocytes, and the mis-sorting of lamellar body cargoes being driven to the lysosomal degradation pathway. Our results highlight the importance of Rab11a-dependent regulation of lamellar body biogenesis in keratinocytes and consequently on epidermal barrier homeostasis. PMID:26872604

  12. Deacetylation of TFEB promotes fibrillar Aβ degradation by upregulating lysosomal biogenesis in microglia.

    PubMed

    Bao, Jintao; Zheng, Liangjun; Zhang, Qi; Li, Xinya; Zhang, Xuefei; Li, Zeyang; Bai, Xue; Zhang, Zhong; Huo, Wei; Zhao, Xuyang; Shang, Shujiang; Wang, Qingsong; Zhang, Chen; Ji, Jianguo

    2016-06-01

    Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD. PMID:27209302

  13. PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation to promote metastasis

    PubMed Central

    LeBleu, Valerie S.; O'Connell, Joyce T.; Herrera, Karina N. Gonzalez; Wikman-Kocher, Harriet; Pantel, Klaus; Haigis, Marcia C.; de Carvalho, Fernanda Machado; Damascena, Aline; Chinen, Ludmilla Thome Domingos; Rocha, Rafael M.; Asara, John M.; Kalluri, Raghu

    2014-01-01

    Cancer cells can divert metabolites into anabolic pathways to support their rapid proliferation and to accumulate the cellular building blocks required for tumor growth. However, the specific bioenergetic profile of invasive and metastatic cancer cells is unknown. Here we report that migratory/invasive cancer cells specifically favor mitochondrial respiration and increased ATP production. Invasive cancer cells use transcription co-activator, PGC-1α to enhance oxidative phosphorylation, mitochondrial biogenesis and oxygen consumption rate. Clinical analysis of human invasive breast cancers revealed a strong correlation between PGC-1α expression in invasive cancer cells and formation of distant metastases. Silencing of PGC-1α in cancer cells suspended their invasive potential and attenuated metastasis without affecting proliferation, primary tumor growth or epithelial-to-mesenchymal (EMT) program. While inherent genetics of cancer cells determine the transcriptome framework required for invasion and metastasis, mitochondrial biogenesis and respiration induced by PGC-1α is also essential for functional motility of cancer cells and metastasis. PMID:25241037

  14. Biogenesis, Function, and Applications of Virus-Derived Small RNAs in Plants

    PubMed Central

    Zhang, Chao; Wu, Zujian; Li, Yi; Wu, Jianguo

    2015-01-01

    RNA silencing, an evolutionarily conserved and sequence-specific gene-inactivation system, has a pivotal role in antiviral defense in most eukaryotic organisms. In plants, a class of exogenous small RNAs (sRNAs) originating from the infecting virus called virus-derived small interfering RNAs (vsiRNAs) are predominantly responsible for RNA silencing-mediated antiviral immunity. Nowadays, the process of vsiRNA formation and the role of vsiRNAs in plant viral defense have been revealed through deep sequencing of sRNAs and diverse genetic analysis. The biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which require diverse essential components including dicer-like (DCL), argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins. vsiRNAs trigger antiviral defense through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) of viral RNA, and they hijack the host RNA silencing system to target complementary host transcripts. Additionally, several applications that take advantage of the current knowledge of vsiRNAs research are being used, such as breeding antiviral plants through genetic engineering technology, reconstructing of viral genomes, and surveying viral ecology and populations. Here, we will provide an overview of vsiRNA pathways, with a primary focus on the advances in vsiRNA biogenesis and function, and discuss their potential applications as well as the future challenges in vsiRNAs research. PMID:26617580

  15. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes.

    PubMed

    Burch, Tanya C; Rhim, Johng S; Nyalwidhe, Julius O

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT(2) PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions. PMID:27478826

  16. Salidroside Stimulates Mitochondrial Biogenesis and Protects against H2O2-Induced Endothelial Dysfunction

    PubMed Central

    Xing, Shasha; Yang, Xiaoyan; Li, Wenjing; Bian, Fang; Wu, Dan; Chi, Jiangyang; Xu, Gao; Zhang, Yonghui; Jin, Si

    2014-01-01

    Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙−) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways. PMID:24868319

  17. Role of membrane glycerolipids in photosynthesis, thylakoid biogenesis and chloroplast development.

    PubMed

    Kobayashi, Koichi

    2016-07-01

    The lipid bilayer of the thylakoid membrane in plant chloroplasts and cyanobacterial cells is predominantly composed of four unique lipid classes; monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). MGDG and DGDG are uncharged galactolipids that constitute the bulk of thylakoid membrane lipids and provide a lipid bilayer matrix for photosynthetic complexes as the main constituents. The glycolipid SQDG and phospholipid PG are anionic lipids with a negative charge on their head groups. SQDG and PG substitute for each other to maintain the amount of total anionic lipids in the thylakoid membrane, with PG having indispensable functions in photosynthesis. In addition to biochemical studies, extensive analyses of mutants deficient in thylakoid lipids have revealed important roles of these lipids in photosynthesis and thylakoid membrane biogenesis. Moreover, recent studies of Arabidopsis thaliana suggest that thylakoid lipid biosynthesis triggers the expression of photosynthesis-associated genes in both the nucleus and plastids and activates the formation of photosynthetic machineries and chloroplast development. Meanwhile, galactolipid biosynthesis is regulated in response to chloroplast functionality and lipid metabolism at transcriptional and post-translational levels. This review summarizes the roles of thylakoid lipids with their biosynthetic pathways in plants and discusses the coordinated regulation of thylakoid lipid biosynthesis with the development of photosynthetic machinery during chloroplast biogenesis. PMID:27114097

  18. Aroma biogenesis and distribution between olive pulps and seeds with identification of aroma trends among cultivars.

    PubMed

    Reboredo-Rodríguez, P; González-Barreiro, C; Cancho-Grande, B; Simal-Gándara, J

    2013-11-01

    The two constitutive parts of four cultivars (Arbequina, Picual, Local and Manzanilla de Sevilla) grown in Spain were separately analysed in order to establish the role of pulp and seed in the biogenesis of extra virgin olive oil (EVOO) aroma through the lipoxygenase (LOX) pathway. C6 and C5 volatile compounds responsible of EVOO aroma were produced by endogenous enzymes in both parts of olive fruits and the differences can be attributed to different enzymes distribution in pulp and seed. According to results, C6 and C5 volatile compounds have mainly their biogenesis in pulp (80-90%) vs. seed (20-10%), independently of the cultivar considered. A linear discriminant analysis was used to establish discriminant aroma compounds between pulp and seed related to the maturity index. A decrease in trans-2-hexen-1-al and an increase in 1-hexanol with ripeness were observed independently of the cultivar considered. Finally, Partial Least Squares (PLS) regression analysis between pulp and seed aroma compounds allowed to establish those volatile compounds that better describe each cultivar. PMID:23768404

  19. Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

    PubMed

    Tanguy, Emeline; Carmon, Ophélie; Wang, Qili; Jeandel, Lydie; Chasserot-Golaz, Sylvette; Montero-Hadjadje, Maité; Vitale, Nicolas

    2016-06-01

    The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015). PMID:26877188

  20. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes

    PubMed Central

    Burch, Tanya C.; Rhim, Johng S.

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT2 PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions. PMID:27478826

  1. Biogenesis, Function, and Applications of Virus-Derived Small RNAs in Plants.

    PubMed

    Zhang, Chao; Wu, Zujian; Li, Yi; Wu, Jianguo

    2015-01-01

    RNA silencing, an evolutionarily conserved and sequence-specific gene-inactivation system, has a pivotal role in antiviral defense in most eukaryotic organisms. In plants, a class of exogenous small RNAs (sRNAs) originating from the infecting virus called virus-derived small interfering RNAs (vsiRNAs) are predominantly responsible for RNA silencing-mediated antiviral immunity. Nowadays, the process of vsiRNA formation and the role of vsiRNAs in plant viral defense have been revealed through deep sequencing of sRNAs and diverse genetic analysis. The biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which require diverse essential components including dicer-like (DCL), argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins. vsiRNAs trigger antiviral defense through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) of viral RNA, and they hijack the host RNA silencing system to target complementary host transcripts. Additionally, several applications that take advantage of the current knowledge of vsiRNAs research are being used, such as breeding antiviral plants through genetic engineering technology, reconstructing of viral genomes, and surveying viral ecology and populations. Here, we will provide an overview of vsiRNA pathways, with a primary focus on the advances in vsiRNA biogenesis and function, and discuss their potential applications as well as the future challenges in vsiRNAs research. PMID:26617580

  2. NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling

    PubMed Central

    Lindström, Mikael S.

    2011-01-01

    At a first glance, ribosome biogenesis and chromatin remodeling are quite different processes, but they share a common problem involving interactions between charged nucleic acids and small basic proteins that may result in unwanted intracellular aggregations. The multifunctional nuclear acidic chaperone NPM1 (B23/nucleophosmin) is active in several stages of ribosome biogenesis, chromatin remodeling, and mitosis as well as in DNA repair, replication and transcription. In addition, NPM1 plays an important role in the Myc-ARF-p53 pathway as well as in SUMO regulation. However, the relative importance of NPM1 in these processes remains unclear. Provided herein is an update on the expanding list of the diverse activities and interacting partners of NPM1. Mechanisms of NPM1 nuclear export functions of NPM1 in the nucleolus and at the mitotic spindle are discussed in relation to tumor development. It is argued that the suggested function of NPM1 as a histone chaperone could explain several, but not all, of the effects observed in cells following changes in NPM1 expression. A future challenge is to understand how NPM1 is activated, recruited, and controlled to carry out its functions. PMID:21152184

  3. Mitochondrial Biogenesis: A Therapeutic Target for Neurodevelopmental Disorders and Neurodegenerative Diseases

    PubMed Central

    Uittenbogaard, Martine; Chiaramello, Anne

    2014-01-01

    In the developing and mature brain, mitochondria act as central hubs for distinct but interwined pathways, necessary for neural development, survival, activity, connectivity and plasticity. In neurons, mitochondria assume diverse functions, such as energy production in the form of ATP, calcium buffering and generation of reactive oxygen species. Mitochondrial dysfunction contributes to a range of neurodevelopmental and neurodegenerative diseases, making mitochondria a potential target for pharmacological-based therapies. Pathogenesis associated with these diseases is accompanied by an increase in mitochondrial mass, a quantitative increase to overcome a qualitative deficiency due to mutated mitochondrial proteins that are either nuclear- or mitochondrial-encoded. This compensatory biological response is maladaptive, as it fails to sufficiently augment the bioenergetically functional mitochondrial mass and correct for the ATP deficit. Since regulation of neuronal mitochondrial biogenesis has been scantily investigated, our current understanding on the network of transcriptional regulators, co-activators and signaling regulators mainly derives from other cellular systems. The purpose of this review is to present the current state of our knowledge and understanding of the transcriptional and signaling cascades controlling neuronal mitochondrial biogenesis and the various therapeutic approaches to enhance the functional mitochondrial mass in the context of neurodevelopmental disorders and adult-onset neurodegenerative diseases. PMID:24606804

  4. Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy

    PubMed Central

    Woldt, Estelle; Sebti, Yasmine; Solt, Laura A.; Duhem, Christian; Lancel, Steve; Eeckhoute, Jérôme; Hesselink, Matthijs K.C.; Paquet, Charlotte; Delhaye, Stéphane; Shin, Youseung; Kamenecka, Theodore M.; Schaart, Gert; Lefebvre, Philippe; Nevière, Rémi; Burris, Thomas P.; Schrauwen, Patrick; Staels, Bart; Duez, Hélène

    2013-01-01

    The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-α is highly expressed in oxidative skeletal muscle and plays a role in mitochondrial biogenesis and oxidative function, in gain- and loss-of function studies. Rev-erb-α-deficiency in skeletal muscle leads to reduced mitochondrial content and oxidative function, resulting in compromised exercise capacity. This phenotype was recapitulated in isolated fibers and in muscle cells upon Rev-erbα knock-down, while Rev-erb-α over-expression increased the number of mitochondria with improved respiratory capacity. Rev-erb-α-deficiency resulted in deactivation of the Stk11–Ampk–Sirt1–Ppargc1-α signaling pathway, whereas autophagy was up-regulated, resulting in both impaired mitochondrial biogenesis and increased clearance. Muscle over-expression or pharmacological activation of Rev-erb-α increased respiration and exercise capacity. This study identifies Rev-erb-α as a pharmacological target which improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function. PMID:23852339

  5. Mitochondrial and lysosomal biogenesis are activated following PINK1/parkin-mediated mitophagy.

    PubMed

    Ivankovic, Davor; Chau, Kai-Yin; Schapira, Anthony H V; Gegg, Matthew E

    2016-01-01

    Impairment of the autophagy-lysosome pathway is implicated with the changes in α-synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH-SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock-down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC-1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy-lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α-synuclein and mitochondrial dysfunction in PD. Damaged mitochondria are degraded by the autophagy-lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing

  6. Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

    PubMed Central

    Sauerwald, Julia; Jores, Tobias; Eisenberg-Bord, Michal; Chuartzman, Silvia Gabriela

    2015-01-01

    A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins. PMID:26149385

  7. Peroxisome Biogenesis Disorders: Biological, Clinical and Pathophysiological Perspectives

    ERIC Educational Resources Information Center

    Braverman, Nancy E.; D'Agostino, Maria Daniela; MacLean, Gillian E.

    2013-01-01

    The peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive disorders in which peroxisome assembly is impaired, leading to multiple peroxisome enzyme deficiencies, complex developmental sequelae and progressive disabilities. Mammalian peroxisome assembly involves the protein products of 16 "PEX" genes;…

  8. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1

    PubMed Central

    Corum, Daniel G.; Tsichlis, Philip N.; Muise-Helmericks, Robin C.

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (∼5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ∼1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.—Corum, D. G., Tsichlis, P. N., Muise-Helmericks, R. C. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1. PMID:24081905

  9. Stress triggers mitochondrial biogenesis to preserve steroidogenesis in Leydig cells.

    PubMed

    Gak, Igor A; Radovic, Sava M; Dukic, Aleksandra R; Janjic, Marija M; Stojkov-Mimic, Natasa J; Kostic, Tatjana S; Andric, Silvana A

    2015-10-01

    Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and β-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies. PMID:26036344

  10. Pyrroloquinoline Quinone Stimulates Mitochondrial Biogenesis through cAMP Response Element-binding Protein Phosphorylation and Increased PGC-1α Expression*

    PubMed Central

    Chowanadisai, Winyoo; Bauerly, Kathryn A.; Tchaparian, Eskouhie; Wong, Alice; Cortopassi, Gino A.; Rucker, Robert B.

    2010-01-01

    Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1–6 cells to 10–30 μm PQQ for 24–48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1α, and increased PGC-1α mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1α or CREB expression. Consistent with activation of the PGC-1α pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction. PMID:19861415

  11. A Pivotal Heme-transfer Reaction Intermediate in Cytochrome c Biogenesis*

    PubMed Central

    Mavridou, Despoina A. I.; Stevens, Julie M.; Mönkemeyer, Leonie; Daltrop, Oliver; di Gleria, Katalin; Kessler, Benedikt M.; Ferguson, Stuart J.; Allen, James W. A.

    2012-01-01

    c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway. PMID:22121193

  12. ER exit sites are physical and functional core autophagosome biogenesis components

    PubMed Central

    Graef, Martin; Friedman, Jonathan R.; Graham, Christopher; Babu, Mohan; Nunnari, Jodi

    2013-01-01

    Autophagy is a central homeostasis and stress response pathway conserved in all eukaryotes. One hallmark of autophagy is the de novo formation of autophagosomes. These double-membrane vesicular structures form around and deliver cargo for degradation by the vacuole/lysosome. Where and how autophagosomes form are outstanding questions. Here we show, using proteomic, cytological, and functional analyses, that autophagosomes are spatially, physically, and functionally linked to endoplasmic reticulum exit sites (ERES), which are specialized regions of the endoplasmic reticulum where COPII transport vesicles are generated. Our data demonstrate that ERES are core autophagosomal biogenesis components whose function is required for the hierarchical assembly of the autophagy machinery immediately downstream of the Atg1 kinase complex at phagophore assembly sites. PMID:23904270

  13. Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated "siderosome".

    PubMed

    Imperi, Francesco; Visca, Paolo

    2013-11-01

    The peptidic siderophore pyoverdine is the primary iron uptake system of fluorescent pseudomonads, and a virulence factor in the opportunistic pathogen Pseudomonas aeruginosa. Pyoverdine biogenesis is a co-ordinate process requiring several precursor-generating enzymes and large nonribosomal peptide synthetases (NRPSs) in the cytoplasm, followed by extracytoplasmic maturation. By using cell fractionation, protein-protein interaction, and in vivo labeling assays we obtained evidence that, in P. aeruginosa, pyoverdine NRPSs assemble with precursor-generating enzymes into a membrane-bound multi-enzymatic complex, for which we propose the name "siderosome". The pyoverdine biogenetic complex represents a novel example of subcellular compartmentalization of a secondary metabolic pathway in prokaryotes. PMID:24042050

  14. Glycogen synthase kinase-3 is involved in regulation of ribosome biogenesis in yeast.

    PubMed

    Yabuki, Yukari; Kodama, Yushi; Katayama, Masako; Sakamoto, Akiko; Kanemaru, Hirofumi; Wan, Kun; Mizuta, Keiko

    2014-01-01

    Secretory defects cause transcriptional repression of both ribosomal proteins and ribosomal RNA genes in Saccharomyces cerevisiae. Rrs1, a trans-acting factor that participates in ribosome biogenesis, is involved in the signaling pathway induced by secretory defects. Here, we found that Rrs1 interacts with two homologs of the glycogen synthase kinase-3 (GSK-3), Rim11, and Mrk1. Rrs1 possesses a repetitive consensus amino acid sequence for phosphorylation by GSK-3, and mutation of this sequence abolished the interaction of Rrs1 with Rim11 and Mrk1. Although this mutation did not affect vegetative cell growth or secretory response, disruption of all four genes encoding GSK-3 homologs, especially Mck1, diminished the transcriptional repression of ribosomal protein genes in response to secretory defects. Among the four GSK-3 kinases, Mck1 appears to be the primary mediator of this response, while the other GSK-3 kinases contribute redundantly. PMID:25035982

  15. The Mechanism of Variegation in immutans Provides Insight into Chloroplast Biogenesis

    PubMed Central

    Foudree, Andrew; Putarjunan, Aarthi; Kambakam, Sekhar; Nolan, Trevor; Fussell, Jenna; Pogorelko, Gennady; Rodermel, Steve

    2012-01-01

    The immutans (im) variegation mutant of Arabidopsis has green and white-sectored leaves due to the absence of fully functional plastid terminal oxidase (PTOX), a plastoquinol oxidase in thylakoid membranes. PTOX appears to be at the nexus of a growing number of biochemical pathways in the plastid, including carotenoid biosynthesis, PSI cyclic electron flow, and chlororespiration. During the early steps of chloroplast biogenesis, PTOX serves as an alternate electron sink and is a prime determinant of the redox poise of the developing photosynthetic apparatus. Whereas a lack of PTOX causes the formation of photooxidized plastids in the white sectors of im, compensating mechanisms allow the green sectors to escape the effects of the mutation. This manuscript provides an update on PTOX, the mechanism of im variegation, and findings about im compensatory mechanisms. PMID:23205022

  16. The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

    PubMed Central

    Santos, Joana A.; Alonso-García, Noelia; Macedo-Ribeiro, Sandra; Pereira, Pedro José Barbosa

    2014-01-01

    Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators. PMID:24847070

  17. A more flexible lipoprotein sorting pathway.

    PubMed

    Chahales, Peter; Thanassi, David G

    2015-05-01

    Lipoprotein biogenesis in Gram-negative bacteria occurs by a conserved pathway, each step of which is considered essential. In contrast to this model, LoVullo and colleagues demonstrate that the N-acyl transferase Lnt is not required in Francisella tularensis or Neisseria gonorrhoeae. This suggests the existence of a more flexible lipoprotein pathway, likely due to a modified Lol transporter complex, and raises the possibility that pathogens may regulate lipoprotein processing to modulate interactions with the host. PMID:25755190

  18. Cell-Specific Transcriptional Profiling of Ciliated Sensory Neurons Reveals Regulators of Behavior and Extracellular Vesicle Biogenesis.

    PubMed

    Wang, Juan; Kaletsky, Rachel; Silva, Malan; Williams, April; Haas, Leonard A; Androwski, Rebecca J; Landis, Jessica N; Patrick, Cory; Rashid, Alina; Santiago-Martinez, Dianaliz; Gravato-Nobre, Maria; Hodgkin, Jonathan; Hall, David H; Murphy, Coleen T; Barr, Maureen M

    2015-12-21

    Cilia and extracellular vesicles (EVs) are signaling organelles [1]. Cilia act as cellular sensory antennae, with defects resulting in human ciliopathies. Cilia both release and bind to EVs [1]. EVs are sub-micron-sized particles released by cells and function in both short- and long-range intercellular communication. In C. elegans and mammals, the autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [2]. A fundamental understanding of EV biology and the relationship between the polycystins, cilia, and EVs is lacking. To define properties of a ciliated EV-releasing cell, we performed RNA-seq on 27 GFP-labeled EV-releasing neurons (EVNs) isolated from adult C. elegans. We identified 335 significantly overrepresented genes, of which 61 were validated by GFP reporters. The EVN transcriptional profile uncovered new pathways controlling EV biogenesis and polycystin signaling and also identified EV cargo, which included an antimicrobial peptide and ASIC channel. Tumor-necrosis-associated factor (TRAF) homologs trf-1 and trf-2 and the p38 mitogen-activated protein kinase (MAPK) pmk-1 acted in polycystin-signaling pathways controlling male mating behaviors. pmk-1 was also required for EV biogenesis, independent of the innate immunity MAPK signaling cascade. This first high-resolution transcriptome profile of a subtype of ciliated sensory neurons isolated from adult animals reveals the functional components of an EVN. PMID:26687621

  19. CTRP9 induces mitochondrial biogenesis and protects high glucose-induced endothelial oxidative damage via AdipoR1 -SIRT1- PGC-1α activation.

    PubMed

    Cheng, Liang; Li, Bin; Chen, Xu; Su, Jie; Wang, Hongbing; Yu, Shiqiang; Zheng, Qijun

    2016-09-01

    Vascular lesions caused by endothelial dysfunction are the most common and serious complication of diabetes. The vasoactive potency of CTRP9 has been reported in our previous study via nitric oxide (NO) production. However, the effect of CTRP9 on vascular endothelial cells remains unknown. This study aimed to investigate the protection role of CTRP9 in the primary aortic vascular endothelial cells and HAECs under high-glucose condition. We found that the aortic vascular endothelial cells isolated from mice fed with a high fat diet generated more ROS production than normal cells, along with decreased mitochondrial biogenesis, which was also found in HAECs treated with high glucose. However, the treatment of CTPR9 significantly reduced ROS production and increased the activities of endogenous antioxidant enzymes, the expression of PGC-1α, NRF1, TFAM, ATP5A1 and SIRT1, and the activity of cytochrome c oxidase, indicating an induction of mitochondrial biogenesis. Furthermore, silencing the expression of SIRT1 in HAECs impeded the effect of CTRP9 on mitochondrial biogenesis, while silencing the expression of AdipoR1 in HAECs reversed the expression of SIRT1 and PGC-1α. Based on these findings, this study showed that CTRP9 might induce mitochondrial biogenesis and protect high glucose-induced endothelial oxidative damage via AdipoR1-SIRT1-PGC-1α signaling pathway. PMID:27349872

  20. Protein translocation and thylakoid biogenesis in cyanobacteria.

    PubMed

    Frain, Kelly M; Gangl, Doris; Jones, Alexander; Zedler, Julie A Z; Robinson, Colin

    2016-03-01

    Cyanobacteria exhibit a complex form of membrane differentiation that sets them apart from most bacteria. Many processes take place in the plasma membrane, but photosynthetic light capture, electron transport and ATP synthesis take place in an abundant internal thylakoid membrane. This review considers how this system of subcellular compartmentalisation is maintained, and how proteins are directed towards the various subcompartments--specifically the plasma membrane, periplasm, thylakoid membrane and thylakoid lumen. The involvement of Sec-, Tat- and signal recognition particle- (SRP)-dependent protein targeting pathways is discussed, together with the possible involvement of a so-called 'spontaneous' pathway for the insertion of membrane proteins, previously characterised for chloroplast thylakoid membrane proteins. An intriguing aspect of cyanobacterial cell biology is that most contain only a single set of genes encoding Sec, Tat and SRP components, yet the proteomes of the plasma and thylakoid membranes are very different. The implications for protein sorting mechanisms are considered. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux. PMID:26341016

  1. Human nucleolar protein Nop52 (RRP1/NNP-1) is involved in site 2 cleavage in internal transcribed spacer 1 of pre-rRNAs at early stages of ribosome biogenesis

    PubMed Central

    Yoshikawa, Harunori; Ishikawa, Hideaki; Izumikawa, Keiichi; Miura, Yutaka; Hayano, Toshiya; Isobe, Toshiaki; Simpson, Richard J.; Takahashi, Nobuhiro

    2015-01-01

    During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S. PMID:25969445

  2. Human nucleolar protein Nop52 (RRP1/NNP-1) is involved in site 2 cleavage in internal transcribed spacer 1 of pre-rRNAs at early stages of ribosome biogenesis.

    PubMed

    Yoshikawa, Harunori; Ishikawa, Hideaki; Izumikawa, Keiichi; Miura, Yutaka; Hayano, Toshiya; Isobe, Toshiaki; Simpson, Richard J; Takahashi, Nobuhiro

    2015-06-23

    During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S. PMID:25969445

  3. Biogenesis of Oxalate in Plant Tissues

    PubMed Central

    Chang, Chi-Cheng; Beevers, Harry

    1968-01-01

    Red beet root discs aerated in potassium phosphate for 2 to 3 days and young spinach leaves actively produce oxalate. A series of labeled compounds was supplied to each of these tissues to determine the extent of conversion to oxalate. Similar results were obtained with the 2 tissues except that in the leaf tissue glyoxylate and glycolate were outstandingly good precursors. Carbon from glucose, acetate, and particularly from some acids of the tricarboxylic acid cycle was recovered in oxalate. Extracts from both tissues were found to contain an enzyme which converts oxaloacetate to oxalate and acetate. The enzyme was partially purified and some of its properties are described. A pathway of oxalate synthesis which does not include glycolate or its oxidase is therefore proposed. PMID:16656975

  4. Upper Tropospheric Ozone Between Latitudes 60S and 60N Derived from Nimbus 7 TOMS/THIR Cloud Slicing

    NASA Technical Reports Server (NTRS)

    Ziemke, Jerald R.; Chandra, Sushil; Bhartia, P. K.

    2002-01-01

    This study evaluates the spatial distributions and seasonal cycles in upper tropospheric ozone (pressure range 200-500 hPa) from low to high latitudes (60S to 60N) derived from the satellite retrieval method called "Cloud Slicing." Cloud Slicing is a unique technique for determining ozone profile information in the troposphere by combining co-located measurements of cloud-top, pressure and above-cloud column ozone. For upper tropospheric ozone, co-located measurements of Nimbus 7 Total Ozone Mapping Spectrometer (TOMS) above-cloud column ozone, and Nimbus 7 Temperature Humidity Infrared Radiometer (THIR) cloud-top pressure during 1979-1984 were incorporated. In the tropics, upper tropospheric ozone shows year-round enhancement in the Atlantic region and evidence of a possible semiannual variability. Upper tropospheric ozone outside the tropics shows greatest abundance in winter and spring seasons in both hemispheres with largest seasonal and largest amounts in the NH. These characteristics are similar to lower stratospheric ozone. Comparisons of upper tropospheric column ozone with both stratospheric ozone and a proxy of lower stratospheric air mass (i.e., tropopause pressure) from National Centers for Environmental Prediction (NCEP) suggest that stratosphere-troposphere exchange (STE) may be a significant source for the seasonal variability of upper tropospheric ozone almost everywhere between 60S and 60N except in low latitudes around 10S to 25N where other sources (e.g., tropospheric transport, biomass burning, aerosol effects, lightning, etc.) may have a greater role.

  5. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.

    PubMed

    Shen, Peter S; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H; Cox, James; Cheng, Yifan; Lambowitz, Alan M; Weissman, Jonathan S; Brandman, Onn; Frost, Adam

    2015-01-01

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails"). PMID:25554787

  6. Biogenesis and Assembly of Eukaryotic Cytochrome c Oxidase Catalytic Core

    PubMed Central

    Soto, Ileana C.; Fontanesi, Flavia; Liu, Jingjing; Barrientos, Antoni

    2011-01-01

    Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human. PMID:21958598

  7. Pex19p, a Farnesylated Protein Essential for Peroxisome Biogenesis

    PubMed Central

    Götte, Klaudia; Girzalsky, Wolfgang; Linkert, Michael; Baumgart, Evelyn; Kammerer, Stefan; Kunau, Wolf-Hubert; Erdmann, Ralf

    1998-01-01

    We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p. PMID:9418908

  8. Regulation of ribosome biogenesis in maize embryonic axes during germination.

    PubMed

    Villa-Hernández, J M; Dinkova, T D; Aguilar-Caballero, R; Rivera-Cabrera, F; Sánchez de Jiménez, E; Pérez-Flores, L J

    2013-10-01

    Ribosome biogenesis is a pre-requisite for cell growth and proliferation; it is however, a highly regulated process that consumes a great quantity of energy. It requires the coordinated production of rRNA, ribosomal proteins and non-ribosomal factors which participate in the processing and mobilization of the new ribosomes. Ribosome biogenesis has been studied in yeast and animals; however, there is little information about this process in plants. The objective of the present work was to study ribosome biogenesis in maize seeds during germination, a stage characterized for its fast growth, and the effect of insulin in this process. Insulin has been reported to accelerate germination and to induce seedling growth. It was observed that among the first events reactivated just after 3 h of imbibition are the rDNA transcription and the pre-rRNA processing and that insulin stimulates both of them (40-230%). The transcript of nucleolin, a protein which regulates rDNA transcription and pre-rRNA processing, is among the messages stored in quiescent dry seeds and it is mobilized into the polysomal fraction during the first hours of imbibition (6 h). In contrast, de novo ribosomal protein synthesis was low during the first hours of imbibition (3 and 6 h) increasing by 60 times in later stages (24 h). Insulin increased this synthesis (75%) at 24 h of imbibition; however, not all ribosomal proteins were similarly regulated. In this regard, an increase in RPS6 and RPL7 protein levels was observed, whereas RPL3 protein levels did not change even though its transcription was induced. Results show that ribosome biogenesis in the first stages of imbibition is carried out with newly synthesized rRNA and ribosomal proteins translated from stored mRNA. PMID:23806421

  9. Abnormal Synaptic Vesicle Biogenesis in Drosophila Synaptogyrin Mutants

    PubMed Central

    Stevens, Robin J.; Akbergenova, Yulia; Jorquera, Ramon A.; Littleton, J. Troy

    2012-01-01

    Sustained neuronal communication relies on the coordinated activity of multiple proteins that regulate synaptic vesicle biogenesis and cycling within the presynaptic terminal. Synaptogyrin and synaptophysin are conserved MARVEL domain-containing transmembrane proteins that are among the most abundant synaptic vesicle constituents, although their role in the synaptic vesicle cycle has remained elusive. To further investigate the function of these proteins, we generated and characterized a synaptogyrin (gyr) null mutant in Drosophila, whose genome encodes a single synaptogyrin isoform and lacks a synaptophysin homolog. We demonstrate that Drosophila synaptogyrin plays a modulatory role in synaptic vesicle biogenesis at larval neuromuscular junctions. Drosophila lacking synaptogyrin are viable and fertile and have no overt deficits in motor function. However, ultrastructural analysis of gyr larvae revealed increased synaptic vesicle diameter and enhanced variability in the size of synaptic vesicles. In addition, the resolution of endocytic cisternae into synaptic vesicles in response to strong stimulation is defective in gyr mutants. Electrophysiological analysis demonstrated an increase in quantal size and a concomitant decrease in quantal content, suggesting functional consequences for transmission caused by the loss of synaptogyrin. Furthermore, high-frequency stimulation resulted in increased facilitation and a delay in recovery from synaptic depression, indicating that synaptic vesicle exo-endocytosis is abnormally regulated during intense stimulation conditions. These results suggest that synaptogyrin modulates the synaptic vesicle exo-endocytic cycle and is required for the proper biogenesis of synaptic vesicles at nerve terminals. PMID:23238721

  10. Bmi1 promotes erythroid development through regulating ribosome biogenesis

    PubMed Central

    Gao, Rui; Chen, Sisi; Kobayashi, Michihiro; Yu, Hao; Zhang, Yingchi; Wan, Yang; Young, Sara K.; Soltis, Anthony; Yu, Ming; Vemula, Sasidhar; Fraenkel, Ernest; Cantor, Alan; Antipin, Yevgeniy; Xu, Yang; Yoder, Mervin C.; Wek, Ronald C.; Ellis, Steven R.; Kapur, Reuben; Zhu, Xiaofan; Liu, Yan

    2015-01-01

    While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in down-regulation of transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including diamond blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells (HSPCs) from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. PMID:25385494

  11. Bmi1 promotes erythroid development through regulating ribosome biogenesis.

    PubMed

    Gao, Rui; Chen, Sisi; Kobayashi, Michihiro; Yu, Hao; Zhang, Yingchi; Wan, Yang; Young, Sara K; Soltis, Anthony; Yu, Ming; Vemula, Sasidhar; Fraenkel, Ernest; Cantor, Alan; Antipin, Yevgeniy; Xu, Yang; Yoder, Mervin C; Wek, Ronald C; Ellis, Steven R; Kapur, Reuben; Zhu, Xiaofan; Liu, Yan

    2015-03-01

    While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in decreased transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including Diamond-Blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. PMID:25385494

  12. The miRNA biogenesis in marine bivalves.

    PubMed

    Rosani, Umberto; Pallavicini, Alberto; Venier, Paola

    2016-01-01

    Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs) guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture species Mytilus galloprovincialis and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves. PMID:26989613

  13. The miRNA biogenesis in marine bivalves

    PubMed Central

    Rosani, Umberto; Pallavicini, Alberto

    2016-01-01

    Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs) guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture species Mytilus galloprovincialis and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves. PMID:26989613

  14. Altered Endosome Biogenesis in Prostate Cancer has Biomarker Potential

    PubMed Central

    Johnson, Ian R D; Parkinson-Lawrence, Emma J; Shandala, Tetyana; Weigert, Roberto; Butler, Lisa M; Brooks, Doug A

    2016-01-01

    Prostate cancer is the second most common form of cancer in males, affecting one in eight men by the time they reach the age of 70. Current diagnostic tests for prostate cancer have significant problems with both false negatives and false positives, necessitating the search for new molecular markers. A recent investigation of endosomal and lysosomal proteins revealed that the critical process of endosomal biogenesis might be altered in prostate cancer. Here, a panel of endosomal markers was evaluated in prostate cancer and non-malignant cells and a significant increase in gene and protein expression was found for early, but not late endosomal proteins. There was also a differential distribution of early endosomes, and altered endosomal traffic and signalling of the transferrin receptors (TFRC and TFR2) in prostate cancer cells. These findings support the concept that endosome biogenesis and function is altered in prostate cancer. Microarray analysis of a clinical cohort confirmed the altered endosomal gene expression observed in cultured prostate cancer cells. Furthermore, in prostate cancer patient tissue specimens, the early endosomal marker and adaptor protein APPL1 showed consistently altered basement membrane histology in the vicinity of tumours and concentrated staining within tumour masses. These novel observations on altered early endosome biogenesis provide a new avenue for prostate cancer biomarker investigation and suggest new methods for the early diagnosis and accurate prognosis of prostate cancer. PMID:25080433

  15. Activation of peroxisome proliferator-activated receptor α induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders.

    PubMed

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J; Sims, Katherine B; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-04-17

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

  16. Regulation of Mitochondrial Respiratory Chain Biogenesis by Estrogens/Estrogen Receptors and Physiological, Pathological and Pharmacological Implications

    PubMed Central

    Chen, Jin-Qiang; Cammarata, Patrick R.; Baines, Christopher P.; Yager, James D.

    2009-01-01

    There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17β-estradiol(E2) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E2-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERα and ERβ and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anticancer drug resistance in human breast cancer cells, neuro-protection for Alzheimer’s disease and Parkinson’s disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area. We also suggest a novel conceptual approach that will contribute to innovative regimines for the prevention or treatment of a wide variety of medical complications based on E2/ER-mediated MRC biogenesis pathway. PMID:19559056

  17. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

    PubMed Central

    Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-01-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  18. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site.

    PubMed

    Pillet, Benjamin; García-Gómez, Juan J; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-10-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  19. Pyrroloquinoline Quinone Biogenesis: Demonstration that PqqE from Klebsiella pneumoniae is a Radical SAM Enzyme†

    PubMed Central

    Wecksler, Stephen R.; Stoll, Stefan; Tran, Ha; Magnusson, Olafur T.; Wu, Shu-pao; King, David; Britt, R. David; Klinman, Judith P.

    2009-01-01

    Biogenesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae requires the expression of six genes (pqqA-F). One of these genes (pqqE) encodes a 43 kDa protein (PqqE) that plays a role in the initial steps in PQQ formation (Veletrop et al. (1995) J. Bacteriol. 177, 5088-5098). PqqE contains two highly conserved cysteine motifs at the N and C-termini, with the N-terminal motif comprised of a consensus sequence of CX3CX2C that is unique to a family of proteins known as radical S-adenosyl-L-methionine (SAM) enzymes (Sofia et al. (2001) Nucleic Acids Res. 29, 1097-1106). PqqE from K. pneumoniae was cloned into E. coli and expressed as the native protein and with an N-terminal His6-tag. Anaerobic expression and purification of the His6-tag PqqE results in an enzyme with a brownish-red hue indicative of Fe-S cluster formation. Spectroscopic and physical analyses indicate that PqqE contains a mixture of Fe-S clusters, with the predominant form of the enzyme containing two [4Fe-4S] clusters. PqqE isolated anaerobically yields active enzyme capable of cleaving SAM to methionine and 5′-deoxyadenosine in an uncoupled reaction (kobs = 0.011 ± 0.001 min-1). In this reaction, the 5′-deoxyadenosyl radical either abstracts a hydrogen atom from a solvent accessible position in the enzyme or obtains a proton and electron from buffer. The putative PQQ substrate PqqA has not yet been shown to be modified by PqqE, implying either that PqqA must be modified before becoming the substrate for PqqE and/or that another protein in the biosynthetic pathway is critical for the initial steps in PQQ biogenesis. PMID:19746930

  20. The Effect of Natural LCAT Mutations on the Biogenesis of HDL.

    PubMed

    Fotakis, Panagiotis; Kuivenhoven, Jan Albert; Dafnis, Eugene; Kardassis, Dimitris; Zannis, Vassilis I

    2015-06-01

    We have investigated how the natural LCAT[T147I] and LCAT[P274S] mutations affect the pathway of biogenesis of HDL. Gene transfer of WT LCAT in LCAT(-/-) mice increased 11.8-fold the plasma cholesterol, whereas the LCAT[T147I] and LCAT[P274S] mutants caused a 5.2- and 2.9-fold increase, respectively. The LCAT[P274S] and the WT LCAT caused a monophasic distribution of cholesterol in the HDL region, whereas the LCAT[T147I] caused a biphasic distribution of cholesterol in the LDL and HDL region. Fractionation of plasma showed that the expression of WT LCAT increased plasma apoE and apoA-IV levels and shifted the distribution of apoA-I to lower densities. The LCAT[T147I] and LCAT[P274S] mutants restored partially apoA-I in the HDL3 fraction and LCAT[T147I] increased apoE in the VLD/IDL/LDL fractions. The in vivo functionality of LCAT was further assessed based on is its ability to correct the aberrant HDL phenotype that was caused by the apoA-I[L159R]FIN mutation. Co-infection of apoA-I(-/-) mice with this apoA-I mutant and either of the two mutant LCAT forms restored only partially the HDL biogenesis defect that was caused by the apoA-I[L159R]FIN and generated a distinct aberrant HDL phenotype. PMID:25948084

  1. Biogenesis of antibiotics-viewing its history and glimpses of the future.

    PubMed

    Spížek, J; Sigler, K; Řezanka, T; Demain, A

    2016-07-01

    This review aims at comparing some historical data with the current situation in the study of biogenesis of natural compounds, antibiotics in the first place. Biogenesis of tetracyclines and cycloheximide and related compounds serves as example. Examples of molecular biological and bioinformatics methods used in the study of antibiotic biogenesis are described both in terms of its historical aspects and the current knowledge. PMID:27188629

  2. Clusterin facilitates stress-induced lipidation of LC3 and autophagosome biogenesis to enhance cancer cell survival

    PubMed Central

    Zhang, Fan; Kumano, Masafumi; Beraldi, Eliana; Fazli, Ladan; Du, Caigan; Moore, Susan; Sorensen, Poul; Zoubeidi, Amina; Gleave, Martin E.

    2014-01-01

    We define stress-induced adaptive survival pathways linking autophagy with the molecular chaperone clusterin (CLU) that function to promote anticancer treatment resistance. During treatment stress, CLU co-localizes with LC3 via an LIR-binding sequence within autophagosome membranes, functioning to facilitate LC3–Atg3 heterocomplex stability and LC3 lipidation, and thereby enhance autophagosome biogenesis and autophagy activation. Stress-induced autophagy is attenuated with CLU silencing in CLU−/− mice and human prostate cancer cells. CLU-enhanced cell survival occurs via autophagy-dependent pathways, and is reduced following autophagy inhibition. Combining CLU inhibition with anticancer treatments attenuates autophagy activation, increases apoptosis and reduces prostate cancer growth. This study defines a novel adaptor protein function for CLU under stress conditions, and highlights how co-targeting CLU and autophagy can amplify proteotoxic stress to delay cancer progression. PMID:25503391

  3. Mitochondrial Biogenesis and Proteome Remodeling Promote One-Carbon Metabolism for T Cell Activation.

    PubMed

    Ron-Harel, Noga; Santos, Daniel; Ghergurovich, Jonathan M; Sage, Peter T; Reddy, Anita; Lovitch, Scott B; Dephoure, Noah; Satterstrom, F Kyle; Sheffer, Michal; Spinelli, Jessica B; Gygi, Steven; Rabinowitz, Joshua D; Sharpe, Arlene H; Haigis, Marcia C

    2016-07-12

    Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival. PMID:27411012

  4. A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

    SciTech Connect

    Hirota, Yuko; Kuronita, Toshio; Fujita, Hideaki; Tanaka, Yoshitaka

    2007-12-07

    Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.

  5. Acute {beta}-adrenergic stimulation does not alter mitochondrial protein synthesis or markers of mitochondrial biogenesis in adult men.

    PubMed

    Robinson, Matthew M; Richards, Jennifer C; Hickey, Matthew S; Moore, Daniel R; Phillips, Stuart M; Bell, Christopher; Miller, Benjamin F

    2010-01-01

    Exercise-induced expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is dramatically inhibited in mice pretreated with a beta-adrenergic receptor (beta-AR) antagonist, suggesting that beta-ARs play an important role in the regulation of skeletal muscle PGC-1alpha expression, and potentially, mitochondrial biogenesis. Accordingly, we hypothesized that acute beta-AR stimulation would induce transcriptional pathways involved in skeletal muscle mitochondrial biogenesis in humans. Whole body protein turnover (WBPT), myofibrillar protein synthesis (MyPS), skeletal muscle mitochondrial protein synthesis (MiPS), and mitochondrial biogenic signaling were determined in samples of vastus lateralis obtained on two separate occasions in 10 young adult males following 1 h of continuous intravenous administration of saline (CON) or a nonspecific beta-AR agonist [isoproterenol (ISO): 12 ng.kg fat free mass(-1).min(-1)], combined with coinfusion of [1,2](13)C-leucine. beta-AR stimulation induced appreciable increases in heart rate and systolic blood pressure (both P < 0.001) but did not affect mitochondrial biogenic signaling (no change in PGC-1alpha, TFAM, NRF-1, NRF-2, COX, or NADHox expression via RT-PCR; P > 0.05). Additionally, MiPS [CON: 0.099 +/- 0.028, ISO: 0.074 +/- 0.046 (mean +/- SD); P > 0.05] and MyPS (CON: 0.059 +/- 0.008, ISO: 0.055 +/- 0.009; P > 0.05), as well as measures of WBPT were unaffected. On the basis of this investigation, we conclude that acute intravenous beta-AR stimulation does not increase mitochondrial protein synthesis or biogenesis signals in skeletal muscle. PMID:19907002

  6. Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

    PubMed Central

    Kim, Young-Kook; Kim, Boseon; Kim, V. Narry

    2016-01-01

    Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research. PMID:26976605

  7. Resistance exercise enhances the molecular signaling of mitochondrial biogenesis induced by endurance exercise in human skeletal muscle.

    PubMed

    Wang, Li; Mascher, Henrik; Psilander, Niklas; Blomstrand, Eva; Sahlin, Kent

    2011-11-01

    Combining endurance and strength training (concurrent training) may change the adaptation compared with single mode training. However, the site of interaction and the mechanisms are unclear. We have investigated the hypothesis that molecular signaling of mitochondrial biogenesis after endurance exercise is impaired by resistance exercise. Ten healthy subjects performed either only endurance exercise (E; 1-h cycling at ∼65% of maximal oxygen uptake), or endurance exercise followed by resistance exercise (ER; 1-h cycling + 6 sets of leg press at 70-80% of 1 repetition maximum) in a randomized cross-over design. Muscle biopsies were obtained before and after exercise (1 and 3 h postcycling). The mRNA of genes related to mitochondrial biogenesis [(peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1)α, PGC-1-related coactivator (PRC)] related coactivator) and substrate regulation (pyruvate dehydrogenase kinase-4) increased after both E and ER, but the mRNA levels were about twofold higher after ER (P < 0.01). Phosphorylation of proteins involved in the signaling cascade of protein synthesis [mammalian target of rapamycin (mTOR), ribosomal S6 kinase 1, and eukaryotic elongation factor 2] was altered after ER but not after E. Moreover, ER induced a larger increase in mRNA of genes associated with positive mTOR signaling (cMyc and Rheb). Phosphorylation of AMP-activated protein kinase, acetyl-CoA carboxylase, and Akt increased similarly at 1 h postcycling (P < 0.01) after both types of exercise. Contrary to our hypothesis, the results demonstrate that ER, performed after E, amplifies the adaptive signaling response of mitochondrial biogenesis compared with single-mode endurance exercise. The mechanism may relate to a cross talk between signaling pathways mediated by mTOR. The results suggest that concurrent training may be beneficial for the adaptation of muscle oxidative capacity. PMID:21836044

  8. Ribosome biogenesis in replicating cells: Integration of experiment and theory.

    PubMed

    Earnest, Tyler M; Cole, John A; Peterson, Joseph R; Hallock, Michael J; Kuhlman, Thomas E; Luthey-Schulten, Zaida

    2016-10-01

    Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016. PMID:27294303

  9. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

    PubMed Central

    Khoshnevis, Sohail; Askenasy, Isabel; Dattolo, Maria D.; Young-Erdos, Crystal L.; Stroupe, M. Elizabeth; Karbstein, Katrin

    2016-01-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  10. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    PubMed

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  11. Biogenesis and Functions of Exosomes and Extracellular Vesicles.

    PubMed

    Dreyer, Florian; Baur, Andreas

    2016-01-01

    Research on extracellular vesicles (EVs) is a new and emerging field that is rapidly growing. Many features of these structures still need to be described and discovered. This concerns their biogenesis, their release and cellular entrance mechanisms, as well as their functions, particularly in vivo. Hence our knowledge on EV is constantly evolving and sometimes changing. In our review we summarize the most important facts of our current knowledge about extracellular vesicles and described some of the assumed functions in the context of cancer and HIV infection. PMID:27317183

  12. Mitochondrial biogenesis during differentiation of Artemia salina cysts.

    PubMed

    Schmitt, H; Grossfeld, H; Littauer, U Z

    1973-09-01

    Mitochondria isolated from cysts of Artemia salina (brine shrimp) were found to be devoid of cristae and to possess a low respiratory capability. Hydration of the cysts induces marked biochemical and morphological changes in the mitochondria. Their biogenesis proceeds in two stages. The first stage is completed within 1 h and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c and perhaps some morphological changes. In the second stage there is an increase in the protein-synthesizing capacity of the mitochondria as well as striking changes in mitochondrial morphology leading to the formation of cristae. PMID:4355924

  13. Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function.

    PubMed

    Dautin, Nathalie

    2010-06-01

    Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins. PMID:22069633

  14. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    PubMed Central

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  15. The biogenesis and emerging roles of circular RNAs.

    PubMed

    Chen, Ling-Ling

    2016-04-01

    Circular RNAs (circRNAs) are produced from precursor mRNA (pre-mRNA) back-splicing of thousands of genes in eukaryotes. Although circRNAs are generally expressed at low levels, recent findings have shed new light on their cell type-specific and tissue-specific expression and on the regulation of their biogenesis. Furthermore, the data indicate that circRNAs shape gene expression by titrating microRNAs, regulating transcription and interfering with splicing, thus effectively expanding the diversity and complexity of eukaryotic transcriptomes. PMID:26908011

  16. Participation of Candida albicans Transcription Factor RLM1 in Cell Wall Biogenesis and Virulence

    PubMed Central

    Delgado-Silva, Yolanda; Vaz, Catarina; Carvalho-Pereira, Joana; Carneiro, Catarina; Nogueira, Eugénia; Correia, Alexandra; Carreto, Laura; Silva, Sónia; Faustino, Augusto; Pais, Célia; Oliveira, Rui; Sampaio, Paula

    2014-01-01

    Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources. PMID:24466000

  17. Histidine Methylation of Yeast Ribosomal Protein Rpl3p Is Required for Proper 60S Subunit Assembly

    PubMed Central

    Al-Hadid, Qais; Roy, Kevin; Munroe, William; Dzialo, Maria C.; Chanfreau, Guillaume F.

    2014-01-01

    Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the large ribosomal subunit and in faithful protein production. PMID:24865971

  18. Mitochondrial biogenesis in the pulmonary vasculature during inhalation lung injury and fibrosis

    EPA Science Inventory

    Cell survival and injury repair is facilitated by mitochondrial biogenesis; however, the role of this process in lung repair is unknown. We evaluated mitochondrial biogenesis in the mouse lung in two injuries that cause acute inflammation and in two that cause chronic inflammatio...

  19. Biogenesis and Function of T Cell-Derived Exosomes

    PubMed Central

    Ventimiglia, Leandro N.; Alonso, Miguel A.

    2016-01-01

    Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins, and nucleic acids) confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes. PMID:27583248

  20. The toxin GraT inhibits ribosome biogenesis.

    PubMed

    Ainelo, Andres; Tamman, Hedvig; Leppik, Margus; Remme, Jaanus; Hõrak, Rita

    2016-05-01

    Most bacteria encode numerous chromosomal toxin-antitoxin (TA) systems that are proposed to contribute to stress tolerance, as they are able to shift the cells to a dormant state. Toxins act on a variety of targets with the majority attacking the translational apparatus. Intriguingly, the toxicity mechanisms of even closely related toxins may differ essentially. Here, we report on a new type of TA toxin that inhibits ribosome biogenesis. GraT of the GraTA system has previously been described in Pseudomonas putida as an unusually moderate toxin at optimal growth temperatures. However, GraT causes a severe growth defect at lower temperatures. Here, we demonstrate that GraT causes the accumulation of free ribosomal subunits. Mapping the rRNA 5' ends reveals incomplete processing of the free subunits and quantification of modified nucleosides shows an underrepresentation of late subunit assembly specific modifications. This indicates that GraT inhibits ribosome subunit assembly. Interestingly, GraT effects can be alleviated by modification of the chaperone DnaK, a known facilitator of late stages in ribosome biogenesis. We show that GraT directly interacts with DnaK and suggest two possible models for the role of this interaction in GraT toxicity. PMID:26833678

  1. Dynamic evolution and biogenesis of small RNAs during sex reversal

    PubMed Central

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-01-01

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis via ovotesis. Biogenesis and expressions of miRNAs were dynamically changed during the reversal. Notably, phylogenetic analysis revealed dynamic expansions of miRNAs in vertebrates and an evolutionary trajectory of conserved miR-17-92 cluster in the Eukarya. We showed that the miR-17-92 cluster in vertebrates was generated through multiple duplications from ancestor miR-92 in invertebrates Tetranychus urticae and Daphnia pulex from the Chelicerata around 580 Mya. Moreover, we identified the sexual regulator Dmrt1 as a direct target of the members miR-19a and -19b in the cluster. These data suggested dynamic biogenesis and expressions of small RNAs during sex reversal and revealed multiple expansions and evolutionary trajectory of miRNAs from invertebrates to vertebrates, which implicate small RNAs in sexual reversal and provide new insight into evolutionary and molecular mechanisms underlying sexual reversal. PMID:25944477

  2. Biogenesis and Function of T Cell-Derived Exosomes.

    PubMed

    Ventimiglia, Leandro N; Alonso, Miguel A

    2016-01-01

    Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins, and nucleic acids) confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes. PMID:27583248

  3. Cytochrome c Oxidase Biogenesis: New levels of Regulation

    PubMed Central

    Fontanesi, Flavia; Soto, Ileana C.; Barrientos, Antoni

    2008-01-01

    Summary Eukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are post-translationally degraded. Recent data obtained in the yeast Saccharomyces cerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed. PMID:18465791

  4. Microalgal lipid droplets: composition, diversity, biogenesis and functions.

    PubMed

    Goold, Hugh; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2015-04-01

    Lipid droplet is the major site of neutral lipid storage in eukaryotic cells, and increasing evidence show its involvement in numerous cellular processes such as lipid homeostasis, signaling, trafficking and inter-organelle communications. Although the biogenesis, structure, and functions of lipid droplets have been well documented for seeds of vascular plants, mammalian adipose tissues, insects and yeasts, relative little is known about lipid droplets in microalgae. Over the past 5 years, the growing interest of microalgae as a platform for biofuel, green chemicals or value-added polyunsaturated fatty acid production has brought algal lipid droplets into spotlight. Studies conducted on the green microalga Chlamydomonas reinhardtii and other model microalgae such as Haematococcus and Nannochloropsis species have led to the identification of proteins associated with lipid droplets, which include putative structural proteins different from plant oleosins and animal perilipins, as well as candidate proteins for lipid biosynthesis, mobilization, trafficking and homeostasis. Biochemical and microscopy studies have also started to shed light on the role of chloroplasts in the biogenesis of lipid droplets in Chlamydomonas. PMID:25433857

  5. Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites.

    PubMed

    Haanstra, Jurgen R; González-Marcano, Eglys B; Gualdrón-López, Melisa; Michels, Paul A M

    2016-05-01

    Peroxisomes of organisms belonging to the protist group Kinetoplastea, which include trypanosomatid parasites of the genera Trypanosoma and Leishmania, are unique in playing a crucial role in glycolysis and other parts of intermediary metabolism. They sequester the majority of the glycolytic enzymes and hence are called glycosomes. Their glycosomal enzyme content can vary strongly, particularly quantitatively, between different trypanosomatid species, and within each species during its life cycle. Turnover of glycosomes by autophagy of redundant ones and biogenesis of a new population of organelles play a pivotal role in the efficient adaptation of the glycosomal metabolic repertoire to the sudden, major nutritional changes encountered during the transitions in their life cycle. The overall mechanism of glycosome biogenesis is similar to that of peroxisomes in other organisms, but the homologous peroxins involved display low sequence conservation as well as variations in motifs mediating crucial protein-protein interactions in the process. The correct compartmentalisation of enzymes is essential for the regulation of the trypanosomatids' metabolism and consequently for their viability. For Trypanosoma brucei it was shown that glycosomes also play a crucial role in its life-cycle regulation: a crucial developmental control switch involves the translocation of a protein phosphatase from the cytosol into the organelles. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative of regulation of enzyme activities as an additional means to adapt the metabolic network to the different environmental conditions encountered. PMID:26384872

  6. Insights into the biogenesis of lysosome-related organelles from the study of the Hermansky-Pudlak syndrome.

    PubMed

    Bonifacino, Juan S

    2004-12-01

    Lysosome-related organelles (LROs) are a family of cell-type-specific organelles that include melanosomes, platelet dense bodies, and cytotoxic T cell granules. The name, LRO, recognizes the fact that all of these organelles contain subsets of lysosomal proteins in addition to cell-type-specific proteins. The recent identification of genetic disorders that cause combined defects in several of these organelles indicates that they share common biogenetic pathways. Studies of one of these disorders, the Hermansky-Pudlak syndrome (HPS), have provided helpful insights into the molecular machinery involved in LRO biogenesis. HPS is a genetically heterogeneous disorder caused by mutations in any of 7 genes in humans and 15 genes in mice. These genes encode subunits of 4 multi-protein complexes named AP-3, BLOC-1, BLOC-2 and BLOC-3, in addition to miscellaneous components of the general protein trafficking machinery. The AP-3 complex is a coat protein involved in vesicle formation and cargo selection in the endosomal-lysosomal system. One of these cargo molecules is the melanosomal enzyme, tyrosinase, the missorting of which may explain the defective melanosomes in AP-3-deficient humans and mice. The function of the BLOC complexes is unknown, although they are thought to mediate either vesicle tethering/fusion or cytoplasmic dispersal of LROs. Further studies of these complexes should contribute to the elucidation of the mechanisms of LRO biogenesis and the pathogenesis of HPS. PMID:15838104

  7. Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome.

    PubMed

    Valenti, Daniela; De Rasmo, Domenico; Signorile, Anna; Rossi, Leonardo; de Bari, Lidia; Scala, Iris; Granese, Barbara; Papa, Sergio; Vacca, Rosa Anna

    2013-04-01

    A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) - a natural polyphenol component of green tea - to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content. In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS. PMID:23291000

  8. Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis

    PubMed Central

    Vourekas, Anastassios; Zheng, Qi; Alexiou, Panagiotis; Maragkakis, Manolis; Kirino, Yohei; Gregory, Brian D.; Mourelatos, Zissimos

    2012-01-01

    Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind piRNAs, a class of small germline RNAs whose biogenesis and functions are still largely elusive. We employed high throughput sequencing after crosslinking and immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrates that Miwi binds spermiogenic mRNAs directly, without utilizing piRNAs as guides, and independent biochemical analyses of testis mRNA-ribonucleoproteins (mRNPs) establishes that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis. PMID:22842725

  9. Estrogen-related receptor alpha and PGC-1-related coactivator constitute a novel complex mediating the biogenesis of functional mitochondria.

    PubMed

    Mirebeau-Prunier, Delphine; Le Pennec, Soazig; Jacques, Caroline; Gueguen, Naig; Poirier, Julie; Malthiery, Yves; Savagner, Frédérique

    2010-02-01

    Mitochondrial biogenesis, which depends on nuclear as well as mitochondrial genes, occurs in response to increased cellular ATP demand. The nuclear transcriptional factors, estrogen-related receptor alpha (ERRalpha) and nuclear respiratory factors 1 and 2, are associated with the coordination of the transcriptional machinery governing mitochondrial biogenesis, whereas coactivators of the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family serve as mediators between the environment and this machinery. In the context of proliferating cells, PGC-1-related coactivator (PRC) is a member of the PGC-1 family, which is known to act in partnership with nuclear respiratory factors, but no functional interference between PRC and ERRalpha has been described so far. We explored three thyroid cell lines, FTC-133, XTC.UC1 and RO 82 W-1, each characterized by a different mitochondrial content, and studied their behavior towards PRC and ERRalpha in terms of respiratory efficiency. Overexpression of PRC and ERRalpha led to increased respiratory chain capacity and mitochondrial mass. The inhibition of ERRalpha decreased cell growth and respiratory chain capacity in all three cell lines. However, the inhibition of PRC and ERRalpha produced a greater effect in the oxidative cell model, decreasing the mitochondrial mass and the phosphorylating respiration, whereas the nonphosphorylating respiration remained unchanged. We therefore hypothesize that the ERRalpha-PRC complex plays a role in arresting the cell cycle through the regulation of oxidative phosphorylation in oxidative cells, and through some other pathway in glycolytic cells. PMID:20067526

  10. Spatio-Temporal Dynamics of Yeast Mitochondrial Biogenesis: Transcriptional and Post-Transcriptional mRNA Oscillatory Modules

    PubMed Central

    Lelandais, Gaëlle; Saint-Georges, Yann; Geneix, Colette; Al-Shikhley, Liza; Dujardin, Geneviève; Jacq, Claude

    2009-01-01

    Examples of metabolic rhythms have recently emerged from studies of budding yeast. High density microarray analyses have produced a remarkably detailed picture of cycling gene expression that could be clustered according to metabolic functions. We developed a model-based approach for the decomposition of expression to analyze these data and to identify functional modules which, expressed sequentially and periodically, contribute to the complex and intricate mitochondrial architecture. This approach revealed that mitochondrial spatio-temporal modules are expressed during periodic spikes and specific cellular localizations, which cover the entire oscillatory period. For instance, assembly factors (32 genes) and translation regulators (47 genes) are expressed earlier than the components of the amino-acid synthesis pathways (31 genes). In addition, we could correlate the expression modules identified with particular post-transcriptional properties. Thus, mRNAs of modules expressed “early” are mostly translated in the vicinity of mitochondria under the control of the Puf3p mRNA-binding protein. This last spatio-temporal module concerns mostly mRNAs coding for basic elements of mitochondrial construction: assembly and regulatory factors. Prediction that unknown genes from this module code for important elements of mitochondrial biogenesis is supported by experimental evidence. More generally, these observations underscore the importance of post-transcriptional processes in mitochondrial biogenesis, highlighting close connections between nuclear transcription and cytoplasmic site-specific translation. PMID:19521515

  11. MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

    PubMed Central

    Rorbach, Joanna; Boesch, Pierre; Gammage, Payam A.; Nicholls, Thomas J. J.; Pearce, Sarah F.; Patel, Dipali; Hauser, Andreas; Perocchi, Fabiana; Minczuk, Michal

    2014-01-01

    Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome. PMID:25009282

  12. Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits.

    PubMed

    Doamekpor, Selom K; Lee, Joong-Won; Hepowit, Nathaniel L; Wu, Cheng; Charenton, Clement; Leonard, Marilyn; Bengtson, Mario H; Rajashankar, Kanagalaghatta R; Sachs, Matthew S; Lima, Christopher D; Joazeiro, Claudio A P

    2016-07-19

    The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1's substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1's intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD's role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation. PMID:27385828

  13. A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits.

    PubMed Central

    Schlatter, Harald; Langer, Thomas; Rosmus, Susann; Onneken, Marie-Luise; Fasold, Hugo

    2002-01-01

    Ribosomal subunits are assembled in the nucleus, and mature 40 S and 60 S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60 S ribosomal subunits involves the use of resealed nuclear envelopes. The export of ribosomal subunits from resealed nuclear envelopes is enhanced by cytoplasmic proteins. Here we present evidence that the export-promoting activity was due to the cytoplasmic 90 kDa heat-shock protein (Hsp90). Isolated, purified Hsp90 vastly enhanced the export of 60 S ribosomal subunits from resealed nuclear envelopes, while inhibition of Hsp90 function, either with the Hsp90-binding drug geldanamycin or with anti-Hsp90 antibodies, resulted in reduced release of 60 S ribosomal subunits. To confirm these findings under in vivo conditions, corresponding experiments were performed with Xenopus oocytes using microinjection techniques; the results obtained confirmed the findings obtained with resealed nuclear envelopes. These findings suggest that Hsp90 facilitates the nuclear export of 60 S ribosomal subunits, probably by chaperoning protein interactions during the export process. PMID:11879195

  14. Removal of copper (II) from aqueous solutions by flotation using polyaluminum chloride silicate (PAX-XL60 S) as coagulant and carbonate ion as activator.

    PubMed

    Ghazy, S E; Mahmoud, I A; Ragab, A H

    2006-01-01

    Flotation is a separation technology for removing toxic heavy metal ions from aqueous solutions. Here a simple and rapid flotation procedure is presented for the removal of copper(II) from aqueous solutions. It is based on the use of polyaluminum chloride silicate (PAX-XL60 S) as coagulant and flocculent, carbonate ion as activator and oleic acid (HOL) as surfactant. Both ion and precipitate flotation are included depending on the solution pH. Ion and precipitate flotation in the aqueous HOL-PAX-XL60 S-Cu2+-CO3(2-) system gave powerful preferential removal of Cu2+ (F -100%) over the HOL-PAX-XL60 S-Cu2+ system containing no CO3(2+) ion (F approximately 86%). The role of CO3(2-) ion is also evident from decreasing the dose of PAX-XL60 S from 700 mg l(-1) to 200 mg l(-1). The other parameters, influencing the flotation process, namely: metal ion, surfactant and PAX-XL60 S concentrations, ionic strength, temperature and foreign ions were examined. Moreover, the procedure was successfully applied to recover Cu2+ ions from different volumes up to 11 and from natural water samples. PMID:16457175

  15. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    PubMed

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. PMID:27329527

  16. A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis

    PubMed Central

    2012-01-01

    Background The overall influence of gene interaction in human disease is unknown. In cystic fibrosis (CF) a single allele of the cystic fibrosis transmembrane conductance regulator (CFTR-ΔF508) accounts for most of the disease. In cell models, CFTR-ΔF508 exhibits defective protein biogenesis and degradation rather than proper trafficking to the plasma membrane where CFTR normally functions. Numerous genes function in the biogenesis of CFTR and influence the fate of CFTR-ΔF508. However it is not known whether genetic variation in such genes contributes to disease severity in patients. Nor is there an easy way to study how numerous gene interactions involving CFTR-ΔF would manifest phenotypically. Methods To gain insight into the function and evolutionary conservation of a gene interaction network that regulates biogenesis of a misfolded ABC transporter, we employed yeast genetics to develop a 'phenomic' model, in which the CFTR-ΔF508-equivalent residue of a yeast homolog is mutated (Yor1-ΔF670), and where the genome is scanned quantitatively for interaction. We first confirmed that Yor1-ΔF undergoes protein misfolding and has reduced half-life, analogous to CFTR-ΔF. Gene interaction was then assessed quantitatively by growth curves for approximately 5,000 double mutants, based on alteration in the dose response to growth inhibition by oligomycin, a toxin extruded from the cell at the plasma membrane by Yor1. Results From a comparative genomic perspective, yeast gene interactions influencing Yor1-ΔF biogenesis were representative of human homologs previously found to modulate processing of CFTR-ΔF in mammalian cells. Additional evolutionarily conserved pathways were implicated by the study, and a ΔF-specific pro-biogenesis function of the recently discovered ER membrane complex (EMC) was evident from the yeast screen. This novel function was validated biochemically by siRNA of an EMC ortholog in a human cell line expressing CFTR-ΔF508. The precision and

  17. An inventory of peroxisomal proteins and pathways in Drosophila melanogaster

    PubMed Central

    Faust, Joseph E.; Verma, Avani; Peng, Chengwei; McNew, James A.

    2012-01-01

    Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). While much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to C. elegans, Drosophila appears to only utilize the peroxisome targeting signal (PTS) type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system. PMID:22758915

  18. Promoting PGC-1α-driven mitochondrial biogenesis is detrimental in pressure-overloaded mouse hearts

    PubMed Central

    Karamanlidis, Georgios; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Lee, Chi Fung

    2014-01-01

    Mitochondrial dysfunction in animal models of heart failure is associated with downregulation of the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α pathway. To test whether PGC-1α is an appropriate therapeutic target for increasing mitochondrial biogenesis and improving function in heart failure, we used a transgenic (TG) mouse model of moderate overexpression of PGC-1α (∼3-fold) in the heart. TG mice had small increases in citrate synthase activity and mitochondria size in the heart without alterations in myocardial energetics or cardiac function at baseline. In vivo dobutamine stress increased fractional shortening in wild-type mice, but this increase was attenuated in TG mice, whereas ex vivo isolated perfused TG hearts demonstrated normal functional and energetic response to high workload challenge. When subjected to pressure overload by transverse aortic constriction (TAC), TG mice displayed a significantly greater acute mortality for both male and female mice; however, long-term survival up to 8 wk was similar between the two groups. TG mice also showed a greater decrease in fractional shortening and a greater increase in left ventricular chamber dimension in response to TAC. Mitochondrial gene expression and citrate synthase activity were mildly increased in TG mice compared with wild-type mice, and this difference was also maintained after TAC. Our data suggest that a moderate level of PGC-1α overexpression in the heart compromises acute survival and does not improve cardiac function during chronic pressure overload in mice. PMID:25172896

  19. Cytochrome c Biogenesis System I: an Intricate Process Catalyzed by a Maturase Supercomplex?

    PubMed Central

    Verissimo, Andreia F.; Daldal, Fevzi

    2014-01-01

    Cytochromes c are ubiquitous heme proteins that are found in most living organisms and are essential for various energy production pathways as well as other cellular processes. Their biosynthesis relies on a complex post-translational process, called cytochrome c biogenesis, responsible for the formation of stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of apocytochromes c heme-binding site (C1XXC2H) cysteine residues. In some organisms this process involves up to nine (CcmABCDEFGHI) membrane proteins working together to achieve heme ligation, designated the Cytochrome c maturation (Ccm)-System I. Here, we review recent findings related to the Ccm-System I found in bacteria, archaea and plant mitochondria, with an emphasis on protein interactions between the Ccm components and their substrates (apocytochrome c and heme). We discuss the possibility that the Ccm proteins may form a multi subunit supercomplex (dubbed “Ccm machine”), and based on the currently available data, we present an updated version of a mechanistic model for Ccm. PMID:24631867

  20. The Deubiquitinating Enzyme UBPY Is Required for Lysosomal Biogenesis and Productive Autophagy in Drosophila

    PubMed Central

    Jacomin, Anne-Claire; Bescond, Amandine; Soleilhac, Emmanuelle; Gallet, Benoît; Schoehn, Guy; Fauvarque, Marie-Odile; Taillebourg, Emmanuel

    2015-01-01

    Autophagy is a catabolic process that delivers cytoplasmic components to the lysosomes. Protein modification by ubiquitination is involved in this pathway: it regulates the stability of autophagy regulators such as BECLIN-1 and it also functions as a tag targeting specific substrates to autophagosomes. In order to identify deubiquitinating enzymes (DUBs) involved in autophagy, we have performed a genetic screen in the Drosophila larval fat body. This screen identified Uch-L3, Usp45, Usp12 and Ubpy. In this paper, we show that Ubpy loss of function results in the accumulation of autophagosomes due to a blockade of the autophagy flux. Furthermore, analysis by electron and confocal microscopy of Ubpy-depleted fat body cells revealed altered lysosomal morphology, indicating that Ubpy inactivation affects lysosomal maintenance and/or biogenesis. Lastly, we have shown that shRNA mediated inactivation of UBPY in HeLa cells affects autophagy in a different way: in UBPY-depleted HeLa cells autophagy is deregulated. PMID:26571504

  1. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. PMID:26667172

  2. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

    PubMed Central

    Farha, Maya A.; Czarny, Tomasz L.; Myers, Cullen L.; Worrall, Liam J.; French, Shawn; Conrady, Deborah G.; Wang, Yang; Oldfield, Eric; Strynadka, Natalie C. J.; Brown, Eric D.

    2015-01-01

    Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery. PMID:26283394

  3. Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

    PubMed Central

    2009-01-01

    Background Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. Results Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. Conclusion These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. PMID:19686593

  4. Deciphering the roles of phosphoinositide lipids in phagolysosome biogenesis

    PubMed Central

    Jeschke, Andreas; Haas, Albert

    2016-01-01

    ABSTRACT Professional phagocytes engulf microbial invaders into plasma membrane-derived phagosomes. These mature into microbicidal phagolysosomes, leading to killing of the ingested microbe. Phagosome maturation involves sequential fusion of the phagosome with early endosomes, late endosomes, and the main degradative compartments in cells, lysosomes. Some bacterial pathogens manipulate the phosphoinositide (PIP) composition of phagosome membranes and are not delivered to phagolysosomes, pointing at a role of PIPs in phagosome maturation. This hypothesis is supported by comprehensive microscopic studies. Recently, cell-free reconstitution of fusion between phagosomes and endo(lyso)somes identified phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 3-phosphate [PI(3)P] as key regulators of phagolysosome biogenesis. Here, we describe the emerging roles of PIPs in phagosome maturation and we present tools to study PIP involvement in phagosome trafficking using intact cells or purified compartments. PMID:27489580

  5. The emerging roles of ribosome biogenesis in craniofacial development.

    PubMed

    Ross, Adam P; Zarbalis, Konstantinos S

    2014-01-01

    Neural crest cells (NCCs) are a transient, migratory cell population, which originates during neurulation at the neural folds and contributes to the majority of tissues, including the mesenchymal structures of the craniofacial skeleton. The deregulation of the complex developmental processes that guide migration, proliferation, and differentiation of NCCs may result in a wide range of pathological conditions grouped together as neurocristopathies. Recently, due to their multipotent properties neural crest stem cells have received considerable attention as a possible source for stem cell based regenerative therapies. This exciting prospect underlines the need to further explore the developmental programs that guide NCC differentiation. This review explores the particular importance of ribosome biogenesis defects in this context since a specific interface between ribosomopathies and neurocristopathies exists as evidenced by disorders such as Treacher-Collins-Franceschetti syndrome (TCS) and Diamond-Blackfan anemia (DBA). PMID:24550838

  6. The Virus-Host Interplay: Biogenesis of +RNA Replication Complexes

    PubMed Central

    Reid, Colleen R.; Airo, Adriana M.; Hobman, Tom C.

    2015-01-01

    Positive-strand RNA (+RNA) viruses are an important group of human and animal pathogens that have significant global health and economic impacts. Notable members include West Nile virus, Dengue virus, Chikungunya, Severe acute respiratory syndrome (SARS) Coronavirus and enteroviruses of the Picornaviridae family.Unfortunately, prophylactic and therapeutic treatments against these pathogens are limited. +RNA viruses have limited coding capacity and thus rely extensively on host factors for successful infection and propagation. A common feature among these viruses is their ability to dramatically modify cellular membranes to serve as platforms for genome replication and assembly of new virions. These viral replication complexes (VRCs) serve two main functions: To increase replication efficiency by concentrating critical factors and to protect the viral genome from host anti-viral systems. This review summarizes current knowledge of critical host factors recruited to or demonstrated to be involved in the biogenesis and stabilization of +RNA virus VRCs. PMID:26287230

  7. β2-adrenoceptor agonists in the regulation of mitochondrial biogenesis

    PubMed Central

    Peterson, Yuri K.; Cameron, Robert B.; Wills, Lauren P.; Trager, Richard E.; Lindsey, Chris C.; Beeson, Craig C.; Schnellmann, Rick G.

    2014-01-01

    The stimulation of mitochondrial biogenesis (MB) via cell surface G-protein coupled receptors is a promising strategy for cell repair and regeneration. Here we report the specificity and chemical rationale of a panel of β2-adrenoceptor agonists with regards to MB. Using primary cultures of renal cells, a diverse panel of β2-adrenoceptor agonists elicited three distinct phenotypes: full MB, partial MB, and non-MB. Full MB compounds had efficacy in the low nanomolar range and represent two chemical scaffolds containing three distinct chemical clusters. Interestingly, the MB phenotype did not correlate with reported receptor affinity or chemical similarity. Chemical clusters were then subjected to pharmacophore modeling creating two models with unique and distinct features, consisting of five conserved amongst full MB compounds were identified. The two discrete pharmacophore models were coalesced into a consensus pharmacophore with four unique features elucidating the spatial and chemical characteristics required to stimulate MB. PMID:23954364

  8. Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production.

    PubMed

    Li, Jian-ying; Ye, Lu-peng; Che, Jia-qian; Song, Jia; You, Zheng-ying; Yun, Ki-chan; Wang, Shao-hua; Zhong, Bo-xiong

    2015-08-01

    The silkworm middle silk gland (MSG) is the sericin synthesis and secretion unique sub-organ. The molecular mechanisms of regulating MSG protein synthesis are largely unknown. Here, we performed shotgun proteomic analysis on the three MSG subsections: the anterior (MSG-A), middle (MSG-M), and posterior (MSG-P) regions. The results showed that more strongly expressed proteins in the MSG-A were involved in multiple processes, such as silk gland development and silk protein protection. The proteins that were highly expressed in the MSG-M were enriched in the ribosome pathway. MSG-P proteins with stronger expression were mainly involved in the oxidative phosphorylation and citrate cycle pathways. These results suggest that the MSG-M is the most active region in the sericin synthesis. Furthermore, comparing the proteome of the MSG with the posterior silk gland (PSG) revealed that the specific and highly expressed proteins in the MSG were primarily involved in the ribosome and aminoacyl-tRNA biosynthesis pathways. These results indicate that silk protein synthesis is much more active as a result of the enhancement of translation-related pathways in the MSG. These results also suggest that enhancing ribosome biogenesis is important to the efficient synthesis of silk proteins. PMID:26051239

  9. Exosome Biogenesis, Regulation, and Function in Viral Infection

    PubMed Central

    Alenquer, Marta; Amorim, Maria João

    2015-01-01

    Exosomes are extracellular vesicles released upon fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. They originate as intraluminal vesicles (ILVs) during the process of MVB formation. Exosomes were shown to contain selectively sorted functional proteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing a role in the physiology of the healthy and diseased organism. Challenges in the field include the identification of mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles and the understanding of the underlying processes directing MVBs for degradation or fusion with the plasma membrane. The investigation into the formation and roles of exosomes in viral infection is in its early years. Although still controversial, exosomes can, in principle, incorporate any functional factor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation. This review initially focuses on the composition and biogenesis of exosomes. It then explores the regulatory mechanisms underlying their biogenesis. Exosomes are part of the endocytic system, which is tightly regulated and able to respond to several stimuli that lead to alterations in the composition of its sub-compartments. We discuss the current knowledge of how these changes affect exosomal release. We then summarize how different viruses exploit specific proteins of endocytic sub-compartments and speculate that it could interfere with exosome function, although no direct link between viral usage of the endocytic system and exosome release has yet been reported. Many recent reports have ascribed functions to exosomes released from cells infected with a variety of animal viruses, including viral spread, host immunity, and manipulation of the microenvironment, which are discussed. Given the ever-growing roles and importance of exosomes in viral infections, understanding what regulates their composition and levels, and

  10. Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus.

    PubMed Central

    Gabbert, K K; Goldman, B S; Kranz, R G

    1997-01-01

    The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments. PMID:9286996

  11. A Time to Reap, a Time to Sow: Mitophagy and Biogenesis in Cardiac Pathophysiology

    PubMed Central

    Andres, Allen M.; Stotland, Aleksandr; Queliconi, Bruno B.; Gottlieb, Roberta A.

    2014-01-01

    Balancing mitophagy and mitochondrial biogenesis is essential for maintaining a healthy population of mitochondria and cellular homeostasis. Coordinated interplay between these two forces that govern mitochondrial turnover plays an important role as an adaptive response against various cellular stresses that can compromise cell survival. Failure to maintain the critical balance between mitophagy and mitochondrial biogenesis or homeostatic turnover of mitochondria results in a population of dysfunctional mitochondria that contribute to various disease processes. In this review we outline the mechanics and relationships between mitophagy and mitochondrial biogenesis, and discuss the implications of a disrupted balance between these two forces, with an emphasis on cardiac physiology. PMID:25444712

  12. Resveratrol Induces Hepatic Mitochondrial Biogenesis Through the Sequential Activation of Nitric Oxide and Carbon Monoxide Production

    PubMed Central

    Kim, Seul-Ki; Joe, Yeonsoo; Zheng, Min; Kim, Hyo Jeong; Yu, Jae-Kyoung; Cho, Gyeong Jae; Chang, Ki Churl; Kim, Hyoung Kyu; Han, Jin; Ryter, Stefan W.

    2014-01-01

    Abstract Aims: Nitric oxide (NO) can induce mitochondrial biogenesis in cultured cells, through increased guanosine 3′,5′-monophosphate (cGMP), and activation of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). We sought to determine the role of NO, heme oxygenase-1 (HO-1), and its reaction product (carbon monoxide [CO]) in the induction of mitochondrial biogenesis by the natural antioxidant resveratrol. Results: S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, induced mitochondrial biogenesis in HepG2 hepatoma cells, and in vivo, through stimulation of PGC-1α. NO-induced mitochondrial biogenesis required cGMP, and was mimicked by the cGMP analogue (8-bromoguanosine 3′,5′-cyclic monophosphate [8-Br-cGMP]). Activation of mitochondrial biogenesis by SNAP required HO-1, as it could be reversed by genetic interference of HO-1; and by treatment with the HO inhibitor tin-protoporphyrin-IX (SnPP) in vitro and in vivo. Cobalt protoporphyrin (CoPP)-IX, an HO-1 inducing agent, stimulated mitochondrial biogenesis in HepG2 cells, which could be reversed by the CO scavenger hemoglobin. Application of CO, using the CO-releasing molecule-3 (CORM-3), stimulated mitochondrial biogenesis in HepG2 cells, in a cGMP-dependent manner. Both CoPP and CORM-3-induced mitochondrial biogenesis required NF-E2-related factor-2 (Nrf2) activation and phosphorylation of Akt. The natural antioxidant resveratrol induced mitochondrial biogenesis in HepG2 cells, in a manner dependent on NO biosynthesis, cGMP synthesis, Nrf2-dependent HO-1 activation, and endogenous CO production. Furthermore, resveratrol preserved mitochondrial biogenesis during lipopolysaccharides-induced hepatic inflammation in vivo. Innovation and Conclusions: The complex interplay between endogenous NO and CO production may underlie the mechanism by which natural antioxidants induce mitochondrial biogenesis. Strategies aimed at improving mitochondrial biogenesis may be used as therapeutics

  13. Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

    PubMed Central

    Larson, Charles L.; Beare, Paul A.; Howe, Dale; Heinzen, Robert A.

    2013-01-01

    Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms. PMID:24248335

  14. SUMO-specific Protease 1 Regulates Mitochondrial Biogenesis through PGC-1α*

    PubMed Central

    Cai, Rong; Yu, Tingting; Huang, Chao; Xia, Xuefeng; Liu, Xiaobing; Gu, Jianmin; Xue, Song; Yeh, Edward T.H.; Cheng, Jinke

    2012-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) is a master regulator of mitochondrial biogenesis in response to changes in the cellular environment, physiological or pathological status of mammals. PGC-1α is known to be modified by SUMO (Small Ubiquitin-like Modifier). However, it is not known whether SUMOylation could affect the function of PGC-1α in mitochondrial biogenesis and that how PGC-1α SUMOylation is regulated. In this study, we have identified the role of Sentrin/SUMO-specific protease 1 (SENP1) as a specific SUMO protease to regulate SUMOylation status of PGC-1α. More importantly, we have also found that SENP1 promotes PGC-1α transcription activity, which is essential for the expression of mitochondrial genes and subsequently mitochondrial biogenesis. Thus, we reveal that the SUMOylation of PGC-1α controlled by SENP1 plays an important role in mitochondrial biogenesis and function. PMID:23152500

  15. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  16. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

    PubMed

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4'-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. PMID:27540631

  17. Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy

    PubMed Central

    Ripolone, Michela; Ronchi, Dario; Violano, Raffaella; Vallejo, Dionis; Fagiolari, Gigliola; Barca, Emanuele; Lucchini, Valeria; Colombo, Irene; Villa, Luisa; Berardinelli, Angela; Balottin, Umberto; Morandi, Lucia; Mora, Marina; Bordoni, Andreina; Fortunato, Francesco; Corti, Stefania; Parisi, Daniela; Toscano, Antonio; Sciacco, Monica; DiMauro, Salvatore; Comi, Giacomo P.; Moggio, Maurizio

    2016-01-01

    , implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors—muscle-specificmyogenic factor 5, myoblast determination 1, and myogenin—were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes. PMID:25844556

  18. Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW), and mechanisms of defective cell growth and proliferation caused by PeBoW deficiency in Arabidopsis

    PubMed Central

    Ahn, Chang Sook; Cho, Hui Kyung; Lee, Du-Hwa; Sim, Hee-Jung; Kim, Sang-Gyu; Pai, Hyun-Sook

    2016-01-01

    The nucleolar protein pescadillo (PES) controls biogenesis of the 60S ribosomal subunit through functional interactions with Block of Proliferation 1 (BOP1) and WD Repeat Domain 12 (WDR12) in plants. In this study, we determined protein characteristics and in planta functions of BOP1 and WDR12, and characterized defects in plant cell growth and proliferation caused by a deficiency of PeBoW (PES-BOP1-WDR12) proteins. Dexamethasone-inducible RNAi of BOP1 and WDR12 caused developmental arrest and premature senescence in Arabidopsis, similar to the phenotype of PES RNAi. Both the N-terminal domain and WD40 repeats of BOP1 and WDR12 were critical for specific associations with 60S/80S ribosomes. In response to nucleolar stress or DNA damage, PeBoW proteins moved from the nucleolus to the nucleoplasm. Kinematic analyses of leaf growth revealed that depletion of PeBoW proteins led to dramatically suppressed cell proliferation, cell expansion, and epidermal pavement cell differentiation. A deficiency in PeBoW proteins resulted in reduced cyclin-dependent kinase Type A activity, causing reduced phosphorylation of histone H1 and retinoblastoma-related (RBR) protein. PeBoW silencing caused rapid transcriptional modulation of cell-cycle genes, including reduction of E2Fa and Cyclin D family genes, and induction of several KRP genes, accompanied by down-regulation of auxin-related genes and up-regulation of jasmonic acid-related genes. Taken together, these results suggest that the PeBoW proteins involved in ribosome biogenesis play a critical role in plant cell growth and survival, and their depletion leads to inhibition of cell-cycle progression, possibly modulated by phytohormone signaling. PMID:27440937

  19. Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW), and mechanisms of defective cell growth and proliferation caused by PeBoW deficiency in Arabidopsis.

    PubMed

    Ahn, Chang Sook; Cho, Hui Kyung; Lee, Du-Hwa; Sim, Hee-Jung; Kim, Sang-Gyu; Pai, Hyun-Sook

    2016-09-01

    The nucleolar protein pescadillo (PES) controls biogenesis of the 60S ribosomal subunit through functional interactions with Block of Proliferation 1 (BOP1) and WD Repeat Domain 12 (WDR12) in plants. In this study, we determined protein characteristics and in planta functions of BOP1 and WDR12, and characterized defects in plant cell growth and proliferation caused by a deficiency of PeBoW (PES-BOP1-WDR12) proteins. Dexamethasone-inducible RNAi of BOP1 and WDR12 caused developmental arrest and premature senescence in Arabidopsis, similar to the phenotype of PES RNAi. Both the N-terminal domain and WD40 repeats of BOP1 and WDR12 were critical for specific associations with 60S/80S ribosomes. In response to nucleolar stress or DNA damage, PeBoW proteins moved from the nucleolus to the nucleoplasm. Kinematic analyses of leaf growth revealed that depletion of PeBoW proteins led to dramatically suppressed cell proliferation, cell expansion, and epidermal pavement cell differentiation. A deficiency in PeBoW proteins resulted in reduced cyclin-dependent kinase Type A activity, causing reduced phosphorylation of histone H1 and retinoblastoma-related (RBR) protein. PeBoW silencing caused rapid transcriptional modulation of cell-cycle genes, including reduction of E2Fa and Cyclin D family genes, and induction of several KRP genes, accompanied by down-regulation of auxin-related genes and up-regulation of jasmonic acid-related genes. Taken together, these results suggest that the PeBoW proteins involved in ribosome biogenesis play a critical role in plant cell growth and survival, and their depletion leads to inhibition of cell-cycle progression, possibly modulated by phytohormone signaling. PMID:27440937

  20. IHG-1 Promotes Mitochondrial Biogenesis by Stabilizing PGC-1α

    PubMed Central

    Hickey, Fionnuala B.; Corcoran, James B.; Docherty, Neil G.; Griffin, Brenda; Bhreathnach, Una; Furlong, Fiona; Martin, Finian; Murphy, Madeline

    2011-01-01

    Increased expression of Induced-by-High-Glucose 1 (IHG-1) associates with tubulointerstitial fibrosis in diabetic nephropathy. IHG-1 amplifies TGF-β1 signaling, but the functions of this highly-conserved protein are not well understood. IHG-1 contains a putative mitochondrial-localization domain, and here we report that IHG-1 is specifically localized to mitochondria. IHG-1 overexpression increased mitochondrial mass and stabilized peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Conversely, inhibition of IHG-1 expression decreased mitochondrial mass, downregulated mitochondrial proteins, and PGC-1α-regulated transcription factors, including nuclear respiratory factor 1 and mitochondrial transcription factor A (TFAM), and reduced activity of the TFAM promoter. In the unilateral ureteral obstruction model, we observed higher PGC-1α protein expression and IHG-1 levels with fibrosis. In a gene-expression database, we noted that renal biopsies of human diabetic nephropathy demonstrated higher expression of genes encoding key mitochondrial proteins, including cytochrome c and manganese superoxide dismutase, compared with control biopsies. In summary, these data suggest that IHG-1 increases mitochondrial biogenesis by promoting PGC-1α-dependent processes, potentially contributing to the pathogenesis of renal fibrosis. PMID:21784897

  1. Regulation of Mitoflash Biogenesis and Signaling by Mitochondrial Dynamics.

    PubMed

    Li, Wenwen; Sun, Tao; Liu, Beibei; Wu, Di; Qi, Wenfeng; Wang, Xianhua; Ma, Qi; Cheng, Heping

    2016-01-01

    Mitochondria are highly dynamic organelles undergoing constant network reorganization and exhibiting stochastic signaling events in the form of mitochondrial flashes (mitoflashes). Here we investigate whether and how mitochondrial network dynamics regulate mitoflash biogenesis and signaling. We found that mitoflash frequency was largely invariant when network fragmentized or redistributed in the absence of mitofusin (Mfn) 1, Mfn2, or Kif5b. However, Opa1 deficiency decreased spontaneous mitoflash frequency due to superimposing changes in respiratory function, whereas mitoflash response to non-metabolic stimulation was unchanged despite network fragmentation. In Drp1- or Mff-deficient cells whose mitochondria hyperfused into a single whole-cell reticulum, the frequency of mitoflashes of regular amplitude and duration was again unaltered, although brief and low-amplitude "miniflashes" emerged because of improved detection ability. As the network reorganized, however, the signal mass of mitoflash signaling was dynamically regulated in accordance with the degree of network connectivity. These findings demonstrate a novel functional role of mitochondrial network dynamics and uncover a magnitude- rather than frequency-modulatory mechanism in the regulation of mitoflash signaling. In addition, our data support a stochastic trigger model for the ignition of mitoflashes. PMID:27623243

  2. Oligodendroglial membrane dynamics in relation to myelin biogenesis.

    PubMed

    Ozgen, Hande; Baron, Wia; Hoekstra, Dick; Kahya, Nicoletta

    2016-09-01

    In the central nervous system, oligodendrocytes synthesize a specialized membrane, the myelin membrane, which enwraps the axons in a multilamellar fashion to provide fast action potential conduction and to ensure axonal integrity. When compared to other membranes, the composition of myelin membranes is unique with its relatively high lipid to protein ratio. Their biogenesis is quite complex and requires a tight regulation of sequential events, which are deregulated in demyelinating diseases such as multiple sclerosis. To devise strategies for remedying such defects, it is crucial to understand molecular mechanisms that underlie myelin assembly and dynamics, including the ability of specific lipids to organize proteins and/or mediate protein-protein interactions in healthy versus diseased myelin membranes. The tight regulation of myelin membrane formation has been widely investigated with classical biochemical and cell biological techniques, both in vitro and in vivo. However, our knowledge about myelin membrane dynamics, such as membrane fluidity in conjunction with the movement/diffusion of proteins and lipids in the membrane and the specificity and role of distinct lipid-protein and protein-protein interactions, is limited. Here, we provide an overview of recent findings about the myelin structure in terms of myelin lipids, proteins and membrane microdomains. To give insight into myelin membrane dynamics, we will particularly highlight the application of model membranes and advanced biophysical techniques, i.e., approaches which clearly provide an added value to insight obtained by classical biochemical techniques. PMID:27141942

  3. Regulation of Senescence by microRNA Biogenesis Factors

    PubMed Central

    Abdelmohsen, Kotb; Srikantan, Subramanya; Kang, Min-Ju; Gorospe, Myriam

    2012-01-01

    Senescence represents a state of indefinite growth arrest in cells that have reached their replicative life span, have become damaged, or express aberrant levels of cancer-related proteins. While senescence is widely considered to represent tumor-suppressive mechanism, the accumulation of senescent cells in tissues of older organisms is believed to underlie age-associated losses in physiologic function and age-related diseases. With the emergence of microRNAs (miRNAs) as a major class of molecular regulators of senescence, we review the transcriptional and post-transcriptional factors that control senescence-associated microRNA biosynthesis. Focusing on their enhancement or repression of senescence, we describe the transcription factors that govern the synthesis of primary (pri-)miRNAs, the proteins that control the nuclear processing of pri-miRNAs into precursor (pre-)miRNAs, including RNA editing enzymes, RNases, and RNA helicases, and the cytoplasmic proteins that affect the final processing of pre-miRNAs into mature miRNAs. We discuss how miRNA biogenesis proteins enhance or repress senescence, and thus influence the senescent phenotype that affects normal tissue function and pathology. PMID:22306790

  4. Lipid partitioning at the nuclear envelope controls membrane biogenesis

    PubMed Central

    Barbosa, Antonio Daniel; Sembongi, Hiroshi; Su, Wen-Min; Abreu, Susana; Reggiori, Fulvio; Carman, George M.; Siniossoglou, Symeon

    2015-01-01

    Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage. PMID:26269581

  5. Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase

    PubMed Central

    Craddock, Christian P; Adams, Nicolette; Bryant, Fiona M; Kurup, Smita; Eastmond, Peter J

    2015-01-01

    Coordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant. PMID:26225871

  6. Activated Type 2 Innate Lymphoid Cells regulate Beige Fat Biogenesis

    PubMed Central

    Lee, Min-Woo; Odegaard, Justin I.; Mukundan, Lata; Qiu, Yifu; Molofsky, Ari B.; Nussbaum, Jesse C.; Yun, Karen; Locksley, Richard M.; Chawla, Ajay

    2014-01-01

    SUMMARY Type 2 innate lymphoid cells (ILC2s), an innate source of the type 2 cytokines interleukin (IL)-5 and -13, participate in the maintenance of tissue homeostasis. Although type 2 immunity is critically important for mediating metabolic adaptations to environmental cold, the functions of ILC2s in beige or brown fat development are poorly defined. We report here that activation of ILC2s by IL-33 is sufficient to promote the growth of functional beige fat in thermoneutral mice. Mechanistically, ILC2 activation results in the proliferation of bipotential adipocyte precursors (APs) and their subsequent commitment to the beige fat lineage. Loss- and gain-of-function studies reveal that ILC2-and eosinophil-derived type 2 cytokines stimulate signaling via the IL-4Rα in PDGFRα+ APs to promote beige fat biogenesis. Together, our results highlight a critical role for ILC2s and type 2 cytokines in the regulation of adipocyte precursor numbers and fate, and as a consequence, adipose tissue homeostasis. PMID:25543153

  7. Chemistry, biogenesis, and biological activities of Cinnamomum zeylanicum.

    PubMed

    Jayaprakasha, G K; Rao, L Jagan Mohan

    2011-07-01

    The genus Cinnamomum comprises of several hundreds of species, which are distributed in Asia and Australia. Cinnamomum zeylanicum, the source of cinnamon bark and leaf oils, is an indigenous tree of Sri Lanka, although most oil now comes from cultivated areas. C. zeylanicum is an important spice and aromatic crop having wide applications in flavoring, perfumery, beverages, and medicines. Volatile oils from different parts of cinnamon such as leaves, bark, fruits, root bark, flowers, and buds have been isolated by hydro distillation/steam distillation and supercritical fluid extraction. The chemical compositions of the volatile oils have been identified by GC and GC-MS. More than 80 compounds were identified from different parts of cinnamon. The leaf oil has a major component called eugenol. Cinnamaldehyde and camphor have been reported to be the major components of volatile oils from stem bark and root bark, respectively. Trans-cinnamyl acetate was found to be the major compound in fruits, flowers, and fruit stalks. These volatile oils were found to exhibit antioxidant, antimicrobial, and antidiabetic activities. C. zeylanicum bark and fruits were found to contain proanthocyandins with doubly linked bis-flavan-3-ol units in the molecule. The present review provides a coherent presentation of scattered literature on the chemistry, biogenesis, and biological activities of cinnamon. PMID:21929331

  8. Autophagy-mediated longevity is modulated by lipoprotein biogenesis

    PubMed Central

    Seah, Nicole E.; de Magalhaes Filho, C. Daniel; Petrashen, Anna P.; Henderson, Hope R.; Laguer, Jade; Gonzalez, Julissa; Dillin, Andrew; Hansen, Malene; Lapierre, Louis R.

    2016-01-01

    ABSTRACT Autophagy-dependent longevity models in C. elegans display altered lipid storage profiles, but the contribution of lipid distribution to life-span extension is not fully understood. Here we report that lipoprotein production, autophagy and lysosomal lipolysis are linked to modulate life span in a conserved fashion. We find that overexpression of the yolk lipoprotein VIT/vitellogenin reduces the life span of long-lived animals by impairing the induction of autophagy-related and lysosomal genes necessary for longevity. Accordingly, reducing vitellogenesis increases life span via induction of autophagy and lysosomal lipolysis. Life-span extension due to reduced vitellogenesis or enhanced lysosomal lipolysis requires nuclear hormone receptors (NHRs) NHR-49 and NHR-80, highlighting novel roles for these NHRs in lysosomal lipid signaling. In dietary-restricted worms and mice, expression of VIT and hepatic APOB (apolipoprotein B), respectively, are significantly reduced, suggesting a conserved longevity mechanism. Altogether, our study demonstrates that lipoprotein biogenesis is an important mechanism that modulates aging by impairing autophagy and lysosomal lipolysis. PMID:26671266

  9. RAL-1 controls multivesicular body biogenesis and exosome secretion

    PubMed Central

    Apaydin, Ahmet; Rodriguez, David; Spiegelhalter, Coralie; Hoff-Yoessle, Sarah; Diem, Maxime; Tak, Saurabh; Lefebvre, Olivier; Schwab, Yannick; Goetz, Jacky G.

    2015-01-01

    Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release. PMID:26459596

  10. Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy

    PubMed Central

    Yamano, Koji; Fogel, Adam I; Wang, Chunxin; van der Bliek, Alexander M; Youle, Richard J

    2014-01-01

    Damaged mitochondria can be selectively eliminated by mitophagy. Although two gene products mutated in Parkinson’s disease, PINK1, and Parkin have been found to play a central role in triggering mitophagy in mammals, how the pre-autophagosomal isolation membrane selectively and accurately engulfs damaged mitochondria remains unclear. In this study, we demonstrate that TBC1D15, a mitochondrial Rab GTPase-activating protein (Rab-GAP), governs autophagosome biogenesis and morphology downstream of Parkin activation. To constrain autophagosome morphogenesis to that of the cargo, TBC1D15 inhibits Rab7 activity and associates with both the mitochondria through binding Fis1 and the isolation membrane through the interactions with LC3/GABARAP family members. Another TBC family member TBC1D17, also participates in mitophagy and forms homodimers and heterodimers with TBC1D15. These results demonstrate that TBC1D15 and TBC1D17 mediate proper autophagic encapsulation of mitochondria by regulating Rab7 activity at the interface between mitochondria and isolation membranes. DOI: http://dx.doi.org/10.7554/eLife.01612.001 PMID:24569479

  11. Heat shock proteins: molecular chaperones of protein biogenesis.

    PubMed Central

    Craig, E A; Gambill, B D; Nelson, R J

    1993-01-01

    Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature. Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins. As a reflection of this role, these Hsps have been referred to as molecular chaperones. Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum. Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding. Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins. The function of the proteolytic system is intertwined with that of molecular chaperones. Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins. The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation. In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins. PMID:8336673

  12. Biogenesis, assembly and turnover of photosystem II units.

    PubMed Central

    Baena-González, Elena; Aro, Eva-Mari

    2002-01-01

    Assembly of photosystem II, a multiprotein complex embedded in the thylakoid membrane, requires stoichiometric production of over 20 protein subunits. Since part of the protein subunits are encoded in the chloroplast genome and part in the nucleus, a signalling network operates between the two genetic compartments in order to prevent wasteful production of proteins. Coordinated synthesis of proteins also takes place among the chloroplast-encoded subunits, thus establishing a hierarchy in the protein components that allows a stepwise building of the complex. In addition to this dependence on assembly partners, other factors such as the developmental stage of the plastid and various photosynthesis-related parameters exert a strict control on the accumulation, membrane targeting and assembly of the PSII subunits. Here, we briefly review recent results on this field obtained with three major approaches: biogenesis of photosystem II during the development of chloroplasts from etioplasts, use of photosystem II-specific mutants and photosystem II turnover during its repair cycle. PMID:12437884

  13. Extracellular Streptomyces lividans vesicles: composition, biogenesis and antimicrobial activity

    PubMed Central

    Schrempf, Hildgund; Merling, Philipp

    2015-01-01

    We selected Streptomyces lividans to elucidate firstly the biogenesis and antimicrobial activities of extracellular vesicles that a filamentous and highly differentiated Gram-positive bacterium produces. Vesicle types range in diameter from 110 to 230 nm and 20 to 60 nm, respectively; they assemble to clusters, and contain lipids and phospholipids allowing their in situ imaging by specific fluorescent dyes. The presence of the identified secondary metabolite undecylprodigiosin provokes red fluorescence of a portion of the heterogeneous vesicle populations facilitating in vivo monitoring. Protuberances containing vesicles generate at tips, and alongside of substrate hyphae, and enumerate during late vegetative growth to droplet-like exudates. Owing to in situ imaging in the presence and absence of a green fluorescent vancomycin derivative, we conclude that protuberances comprising vesicles arise at sites with enhanced levels of peptidoglycan subunits [pentapeptide of lipid II (C55)-linked disaccharides], and reduced levels of polymerized and cross-linked peptidoglycan within hyphae. These sites correlate with enhanced levels of anionic phospholipids and lipids. Vesicles provoke pronounced damages of Aspergillus proliferans, Verticillium dahliae and induced clumping and distortion of Escherichia coli. These harmful effects are likely attributable to the action of the identified vesicular compounds including different enzyme types, components of signal transduction cascades and undecylprodigiosin. Based on our pioneering findings, we highlight novel clues with environmental implications and application potential. PMID:25851532

  14. Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori.

    PubMed

    Liechti, George; Goldberg, Joanna B

    2012-01-01

    The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins (OMPs) are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε proteobacteria, while the inner and OM associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual

  15. Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1α transcription and mitochondrial biogenesis in muscle cells

    PubMed Central

    Wu, Zhidan; Huang, Xueming; Feng, Yajun; Handschin, Christoph; Feng, Yan; Gullicksen, P. Scott; Bare, Olivia; Labow, Mark; Spiegelman, Bruce; Stevenson, Susan C.

    2006-01-01

    PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) is a master regulator of mitochondrial biogenesis and plays an important role in several other aspects of energy metabolism. To identify upstream regulators of PGC-1α gene transcription, 10,000 human full-length cDNAs were screened for induction of the PGC-1α promoter. A number of activators of PGC-1α transcription were found; the most potent activator was the transducer of regulated CREB (cAMP response element-binding protein) binding protein (TORC) 1, a coactivator of CREB. The other two members of the TORC family, TORC2 and TORC3, also strongly activated PGC-1α transcription. TORCs dramatically induced PGC-1α gene transcription through CREB. Forced expression of TORCs in primary muscle cells induced the endogenous mRNA of PGC-1α and its downstream target genes in the mitochondrial respiratory chain and TCA cycle. Importantly, these changes in gene expression resulted in increased mitochondrial oxidative capacity measured by cellular respiration and fatty acid oxidation. Finally, we demonstrated that the action of TORCs in promoting mitochondrial gene expression and function requires PGC-1α. Previous studies had indicated that TORCs function as a calcium- and cAMP-sensitive coincidence detector and mediate individual and synergistic effects of these two pathways. Our results, together with previous findings, strongly suggest that TORCs play a key role in linking these external signals to the transcriptional program of adaptive mitochondrial biogenesis by activating PGC-1α gene transcription. PMID:16980408

  16. [RNA interference: biogenesis molecular mechanisms and its applications in cervical cancer].

    PubMed

    Peralta-Zaragoza, Oscar; Bermúdez-Morales, Víctor Hugo; Madrid-Marina, Vicente

    2010-01-01

    RNAi (RNA interference) is a natural process by which eukaryotic cells silence gene expression through small interference RNAs (siRNA) which are complementary to messenger RNA (mRNA). In this process, the siRNA that are 21-25 nucleotides long and are known as microRNA (miRNA), either associate with the RNA-induced silencing complex (RISC), which targets and cleaves the complementary mRNAs by the endonucleolytic pathway, or repress the translation. It is also possible to silence exogenous gene expression during viral infections by using DNA templates to transcribe siRNA with properties that are identical to those of bioactive microRNA. Persistent human papillomavirus (HPV) infection is the main etiological agent during cervical cancer development and the HPV E6 and E7 oncogenes, which induce cellular transformation and immortalization, represent strategic targets to be silenced with siRNA. In several in vitro and in vivo studies, it has been demonstrated that the introduction of siRNA directed against the E6 and E7 oncogenes in human tumoral cervical cells transformed by HPV, leads to the efficient silencing of HPV E6 and E7 oncogene expression, which induces the accumulation of the products of the p53 and pRb tumor suppressor genes and activates the mechanism of programmed cell death by apoptosis; thus, the progression of the tumoral growth process may be prevented. The goal of this review is to analyze the microRNA biogenesis process in the silencing of gene expression and to discuss the different protocols for the use of siRNA as a potential gene therapy strategy for the treatment of cervical cancer. PMID:20415061

  17. Hexosamine and cell wall biogenesis in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Maia, J C

    1994-08-01

    Chitin, a beta-(1-->4) polymer of N-acetyl-glucosamine, is an important constituent of fungal cell walls. This polymer is synthesized by the incorporation of N-acetyl-D-glucosamine units from the precursor UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) in a reaction catalyzed by chitin synthase. In the aquatic fungus Blastocladiella emersonii, chitin, the major component of the cell wall, is synthesized and incorporated in the cell surface of the free-swimming zoospore during the abrupt transition from this wall-less cell to the sessile, wall-containing cyst. Studies with cycloheximide indicate that chitin synthesis occurs in the apparent absence of protein synthesis, and thus posttranslational controls presumably regulate the cell wall biogenesis during encystment. Glutamine: fructose 6-phosphate amidotransferase, first enzyme of the hexosamine biosynthetic pathway, was found to play a central role in the regulation of chitin synthesis in this fungus. This enzyme exists in two forms, which are interconvertible by phosphorylation or dephosphorylation of serine residues. It is allosterically inhibited in the phosphorylated form, as it is in the zoospore, by UDP-GlcNAc. In addition, UDP-GlcNAc inhibits the dephosphorylation of amidotransferase catalyzed by protein phosphatases 2A and 2C. Thus, UDP-GlcNAc plays a dual role in hexosamine and chitin synthesis in zoospore: it not only inhibits the phosphorylated form of the enzyme but also prevents its dephosphorylation. The available data suggest that substrate availability plays a role in the control of chitin synthesis during zoospore differentiation. PMID:8070634

  18. Δ1-Pyrroline-5-Carboxylate/Glutamate Biogenesis Is Required for Fungal Virulence and Sporulation

    PubMed Central

    Yao, Ziting; Zou, Chengwu; Zhou, Hui; Wang, Jinzi; Lu, Lidan; Li, Yang; Chen, Baoshan

    2013-01-01

    Proline dehydrogenase (Prodh) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5Cdh) are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ1-pyrroline-5-carboxylate (P5C) content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica. PMID:24039956

  19. Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs.

    PubMed

    Donaire, Livia; Barajas, Daniel; Martínez-García, Belén; Martínez-Priego, Llucia; Pagán, Israel; Llave, César

    2008-06-01

    In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3' end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities. PMID:18353962

  20. Biochemical pathways in seed oil synthesis.

    PubMed

    Bates, Philip D; Stymne, Sten; Ohlrogge, John

    2013-06-01

    Oil produced in plant seeds is utilized as a major source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. The biochemical pathways leading to oil (triacylglycerol) synthesis in seeds involve multiple subcellular organelles, requiring extensive lipid trafficking. Phosphatidylcholine plays a central role in these pathways as a substrate for acyl modifications and likely as a carrier for the trafficking of acyl groups between organelles and membrane subdomains. Although much has been clarified regarding the enzymes and pathways responsible for acyl-group flux, there are still major gaps in our understanding. These include the identity of several key enzymes, how flux between alternative pathways is controlled and the specialized cell biology leading to biogenesis of oil bodies that store up to 80% of carbon in seeds. PMID:23529069

  1. Autophagy plays a role in skeletal muscle mitochondrial biogenesis in an endurance exercise-trained condition.

    PubMed

    Ju, Jeong-Sun; Jeon, Sei-Il; Park, Je-Young; Lee, Jong-Young; Lee, Seong-Cheol; Cho, Ki-Jung; Jeong, Jong-Moon

    2016-09-01

    Mitochondrial homeostasis is tightly regulated by two major processes: mitochondrial biogenesis and mitochondrial degradation by autophagy (mitophagy). Research in mitochondrial biogenesis in skeletal muscle in response to endurance exercise training has been well established, while the mechanisms regulating mitophagy and the interplay between mitochondrial biogenesis and degradation following endurance exercise training are not yet well defined. The purpose of this study was to examine the effects of a short-term inhibition of autophagy in response to acute endurance exercise on skeletal muscle mitochondrial biogenesis and dynamics in an exercise-trained condition. Male wild-type C57BL/6 mice performed five daily bouts of 1-h swimming per week for 8 weeks. In order to measure autophagy flux in mouse skeletal muscle, mice were treated with or without 2 days of 0.4 mg/kg/day intraperitoneal colchicine (blocking the degradation of autophagosomes) following swimming exercise training. The autophagic flux assay demonstrated that swimming training resulted in an increase in the autophagic flux (~100 % increase in LC3-II) in mouse skeletal muscle. Mitochondrial fusion proteins, Opa1 and MFN2, were significantly elevated, and mitochondrial fission protein, Drp1, was also increased in trained mouse skeletal muscle, suggesting that endurance exercise training promotes both mitochondrial fusion and fission processes. A mitochondrial receptor, Bnip3, was further increased in exercised muscle when treated with colchicine while Pink/Parkin protein levels were unchanged. The endurance exercise training induced increases in mitochondrial biogenesis marker proteins, SDH, COX IV, and a mitochondrial biogenesis promoting factor, PGC-1α but this effect was abolished in colchicine-treated mouse skeletal muscle. This suggests that autophagy plays an important role in mitochondrial biogenesis and this coordination between these opposing processes is involved in the cellular

  2. Expression, covariation, and genetic regulation of miRNA Biogenesis genes in brain supports their role in addiction, psychiatric disorders, and disease

    PubMed Central

    Mulligan, Megan K.; DuBose, Candice; Yue, Junming; Miles, Michael F.; Lu, Lu; Hamre, Kristin M.

    2013-01-01

    The role of miRNA and miRNA biogenesis genes in the adult brain is just beginning to be explored. In this study we have performed a comprehensive analysis of the expression, genetic regulation, and co-expression of major components of the miRNA biogenesis pathway using human and mouse data sets and resources available on the GeneNetwork web site (genenetwork.org). We found a wide range of variation in expression in both species for key components of the pathway—Drosha, Pasha, and Dicer. Across species, tissues, and expression platforms all three genes are generally well-correlated. No single genetic locus exerts a strong and consistent influence on the expression of these key genes across murine brain regions. However, in mouse striatum, many members of the miRNA pathway are correlated—including Dicer, Drosha, Pasha, Ars2 (Srrt), Eif2c1 (Ago1), Eif2c2 (Ago2), Zcchc11, and Snip1. The expression of these genes may be partly influenced by a locus on Chromosome 9 (105.67–106.32 Mb). We explored ~1500 brain phenotypes available for the C57BL/6J × DBA/2J (BXD) genetic mouse population in order to identify miRNA biogenesis genes correlated with traits related to addiction and psychiatric disorders. We found a significant association between expression of Dicer and Drosha in several brain regions and the response to many drugs of abuse, including ethanol, cocaine, and methamphetamine. Expression of Dicer, Drosha, and Pasha in most of the brain regions explored is strongly correlated with the expression of key members of the dopamine system. Drosha, Pasha, and Dicer expression is also correlated with the expression of behavioral traits measuring depression and sensorimotor gating, impulsivity, and anxiety, respectively. Our study provides a global survey of the expression and regulation of key miRNA biogenesis genes in brain and provides preliminary support for the involvement of these genes and their product miRNAs in addiction and psychiatric disease processes

  3. Hyperglycemia decreases mitochondrial function: The regulatory role of mitochondrial biogenesis

    SciTech Connect

    Palmeira, Carlos M. Rolo, Anabela P.; Berthiaume, Jessica; Bjork, James A.; Wallace, Kendall B.

    2007-12-01

    Increased generation of reactive oxygen species (ROS) is implicated in 'glucose toxicity' in diabetes. However, little is known about the action of glucose on the expression of transcription factors in hepatocytes, especially those involved in mitochondrial DNA (mtDNA) replication and transcription. Since mitochondrial functional capacity is dynamically regulated, we hypothesized that stressful conditions of hyperglycemia induce adaptations in the transcriptional control of cellular energy metabolism, including inhibition of mitochondrial biogenesis and oxidative metabolism. Cell viability, mitochondrial respiration, ROS generation and oxidized proteins were determined in HepG2 cells cultured in the presence of either 5.5 mM (control) or 30 mM glucose (high glucose) for 48 h, 96 h and 7 days. Additionally, mtDNA abundance, plasminogen activator inhibitor-1 (PAI-1), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) transcripts were evaluated by real time PCR. High glucose induced a progressive increase in ROS generation and accumulation of oxidized proteins, with no changes in cell viability. Increased expression of PAI-1 was observed as early as 96 h of exposure to high glucose. After 7 days in hyperglycemia, HepG2 cells exhibited inhibited uncoupled respiration and decreased MitoTracker Red fluorescence associated with a 25% decrease in mtDNA and 16% decrease in TFAM transcripts. These results indicate that glucose may regulate mtDNA copy number by modulating the transcriptional activity of TFAM in response to hyperglycemia-induced ROS production. The decrease of mtDNA content and inhibition of mitochondrial function may be pathogenic hallmarks in the altered metabolic status associated with diabetes.

  4. Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

    PubMed Central

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2008-01-01

    Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1− Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1− cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. PMID:18550798

  5. A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

    PubMed

    Feiz, Leila; Williams-Carrier, Rosalind; Belcher, Susan; Montano, Monica; Barkan, Alice; Stern, David B

    2014-12-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. PMID:25279696

  6. Expression profiles of respiratory components associated with mitochondrial biogenesis during germination and seedling growth under normal and restricted conditions in wheat.

    PubMed

    Naydenov, Nayden G; Khanam, Sakina M; Atanassov, Atanas; Nakamura, Chiharu

    2008-02-01

    Germination of plant embryo is a dynamic phase-changing process that is driven by a rapid increase in mitochondrial respiration. We studied the development of respiratory electron transport pathways and the profiles of their transcript and protein components during this critical period using wheat embryos. Oxygen consumption through both the cytochrome and alternative pathways increased rapidly upon imbibition. Quantitative RT-PCR analysis using specific primers and western blot analysis using specific antibodies suggested that this respiratory burst was supported both by the stored mRNA and protein components and ones synthesized de novo at least in the cytochrome pathway. Dry embryos also contained transcript and protein of alternative oxidase (AOX), but their levels remained constant during the studied period. By contrast, the alternative pathway capacity showed a marked increase when the cytochrome pathway was inhibited by antimycin A and this increase was associated with increased levels of AOX transcript and protein. Our results suggest that mitochondrial biogenesis is accompanied by sequential and differential gene expression and protein accumulation, and that AOX allows the complex I to continue to conserve energy thus to support embryo germination and initial seedling growth in wheat when the cytochrome pathway is restricted. PMID:18379132

  7. RNA Binding Proteins in the miRNA Pathway

    PubMed Central

    Connerty, Patrick; Ahadi, Alireza; Hutvagner, Gyorgy

    2015-01-01

    microRNAs (miRNAs) are short ~22 nucleotides (nt) ribonucleic acids which post-transcriptionally regulate gene expression. miRNAs are key regulators of all cellular processes, and the correct expression of miRNAs in an organism is crucial for proper development and cellular function. As a result, the miRNA biogenesis pathway is highly regulated. In this review, we outline the basic steps of miRNA biogenesis and miRNA mediated gene regulation focusing on the role of RNA binding proteins (RBPs). We also describe multiple mechanisms that regulate the canonical miRNA pathway, which depends on a wide range of RBPs. Moreover, we hypothesise that the interaction between miRNA regulation and RBPs is potentially more widespread based on the analysis of available high-throughput datasets. PMID:26712751

  8. A liaison between mTOR signaling, ribosome biogenesis and cancer☆

    PubMed Central

    Gentilella, Antonio; Kozma, Sara C.; Thomas, George

    2016-01-01

    The ability to translate genetic information into functional proteins is considered a landmark in evolution. Ribosomes have evolved to take on this responsibility and, although there are some differences in their molecular make-up, both prokaryotes and eukaryotes share a common structural architecture and similar underlying mechanisms of protein synthesis. Understanding ribosome function and biogenesis has been the focus of extensive research since the early days of their discovery. In the last decade however, new and unexpected roles have emerged that place deregulated ribosome biogenesis and protein synthesis at the crossroads of pathological settings, particularly cancer, revealing a set of novel cellular checkpoints. Moreover, it is also becoming evident that mTOR signaling, which regulates an array of anabolic processes, including ribosome biogenesis, is often exploited by cancer cells to sustain proliferation through the upregulation of global protein synthesis. The use of pharmacological agents that interfere with ribosome biogenesis and mTOR signaling has proven to be an effective strategy to control cancer development clinically. Here we discuss the most recent findings concerning the underlying mechanisms by which mTOR signaling controls ribosome production and the potential impact of ribosome bio-genesis in tumor development. This article is part of a Special Issue entitled: Translation and Cancer. PMID:25735853

  9. Eukaryote-specific rRNA expansion segments function in ribosome biogenesis.

    PubMed

    Ramesh, Madhumitha; Woolford, John L

    2016-08-01

    The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis. PMID:27317789

  10. Echinochrome A Increases Mitochondrial Mass and Function by Modulating Mitochondrial Biogenesis Regulatory Genes

    PubMed Central

    Jeong, Seung Hun; Kim, Hyoung Kyu; Song, In-Sung; Noh, Su Jin; Marquez, Jubert; Ko, Kyung Soo; Rhee, Byoung Doo; Kim, Nari; Mishchenko, Natalia P.; Fedoreyev, Sergey A.; Stonik, Valentin A.; Han, Jin

    2014-01-01

    Echinochrome A (Ech A) is a natural pigment from sea urchins that has been reported to have antioxidant properties and a cardio protective effect against ischemia reperfusion injury. In this study, we ascertained whether Ech A enhances the mitochondrial biogenesis and oxidative phosphorylation in rat cardio myoblast H9c2 cells. To study the effects of Ech A on mitochondrial biogenesis, we measured mitochondrial mass, level of oxidative phosphorylation, and mitochondrial biogenesis regulatory gene expression. Ech A treatment did not induce cytotoxicity. However, Ech A treatment enhanced oxygen consumption rate and mitochondrial ATP level. Likewise, Ech A treatment increased mitochondrial contents in H9c2 cells. Furthermore, Ech A treatment up-regulated biogenesis of regulatory transcription genes, including proliferator-activated receptor gamma co-activator (PGC)-1α, estrogen-related receptor (ERR)-α, peroxisome proliferator-activator receptor (PPAR)-γ, and nuclear respiratory factor (NRF)-1 and such mitochondrial transcription regulatory genes as mitochondrial transcriptional factor A (TFAM), mitochondrial transcription factor B2 (TFB2M), mitochondrial DNA direct polymerase (POLMRT), single strand binding protein (SSBP) and Tu translation elongation factor (TUFM). In conclusion, these data suggest that Ech A is a potentiated marine drug which enhances mitochondrial biogenesis. PMID:25196935

  11. Rhodobacter capsulatus CycH: a bipartite gene product with pleiotropic effects on the biogenesis of structurally different c-type cytochromes.

    PubMed Central

    Lang, S E; Jenney, F E; Daldal, F

    1996-01-01

    While searching for components of the soluble electron carrier (cytochrome c2)-independent photosynthetic (Ps) growth pathway in Rhodobacter capsulatus, a Ps- mutant (FJM13) was isolated from a Ps+ cytochrome c2-strain. This mutant could be complemented to Ps+ growth by cycA encoding the soluble cytochrome c2 but was unable to produce several c-type cytochromes. Only cytochrome c1 of the cytochrome bc1 complex was present in FJM13 cells grown on enriched medium, while cells grown on minimal medium contained at various levels all c-type cytochromes, including the membrane-bound electron carrier cytochrome cy. Complementation of FJM13 by a chromosomal library lacking cycA yielded a DNA fragment which also complemented a previously described Ps- mutant, MT113, known to lack all c-type cytochromes. Deletion and DNA sequence analyses revealed an open reading frame homologous to cycH, involved in cytochrome c biogenesis. The cycH gene product (CycH) is predicted to be a bipartite protein with membrane-associated amino-terminal (CycH1) and periplasmic carboxyl-terminal (CycH2) subdomains. Mutations eliminating CyCH drastically decrease the production or all known c-type cytochromes. However, mutations truncating only its CycH2 subdomain always produce cytochrome c1 and affect the presence of other cytochromes to different degrees in a growth medium-dependent manner. Thus, the subdomain CycH1 is sufficient for the proper maturation of cytochrome c1 which is the only known c-type cytochrome anchored to the cytoplasmic membrane by its carboxyl terminus, while CycH2 is required for efficient biogenesis of other c-type cytochromes. These findings demonstrate that the two subdomains of CycH play different roles in the biogenesis of topologically distinct c-type cytochromes and reconcile the apparently conflicting data previously obtained for other species. PMID:8752349

  12. Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing

    PubMed Central

    Le Thomas, Adrien; Stuwe, Evelyn; Li, Sisi; Marinov, Georgi; Rozhkov, Nikolay; Chen, Yung-Chia Ariel; Luo, Yicheng; Sachidanandam, Ravi; Toth, Katalin Fejes; Patel, Dinshaw; Aravin, Alexei A.

    2014-01-01

    Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. PMID:25085419

  13. MTR4, a putative RNA helicase and exosome co-factor, is required for proper rRNA biogenesis and development in Arabidopsis thaliana.

    PubMed

    Lange, Heike; Sement, François M; Gagliardi, Dominique

    2011-10-01

    The exosome is a conserved protein complex that is responsible for essential 3'→5' RNA degradation in both the nucleus and the cytosol. It is composed of a nine-subunit core complex to which co-factors confer both RNA substrate recognition and ribonucleolytic activities. Very few exosome co-factors have been identified in plants. Here, we have characterized a putative RNA helicase, AtMTR4, that is involved in the degradation of several nucleolar exosome substrates in Arabidopsis thaliana. We show that AtMTR4, rather than its closely related protein HEN2, is required for proper rRNA biogenesis in Arabidopsis. AtMTR4 is mostly localized in the nucleolus, a subcellular compartmentalization that is shared with another exosome co-factor, RRP6L2. AtMTR4 and RRP6L2 cooperate in several steps of rRNA maturation and surveillance, such as processing the 5.8S rRNA and removal of rRNA maturation by-products. Interestingly, degradation of the Arabidopsis 5' external transcribed spacer (5' ETS) requires cooperation of both the 5'→3' and 3'→5' exoribonucleolytic pathways. Accumulating AtMTR4 targets give rise to illegitimate small RNAs; however, these do not affect rRNA metabolism or contribute to the phenotype of mtr4 mutants. Plants lacking AtMTR4 are viable but show several developmental defects, including aberrant vein patterning and pointed first leaves. The mtr4 phenotype resembles that of several ribosomal protein and nucleolin mutants, and may be explained by delayed ribosome biogenesis, as we observed a reduced rate of rRNA accumulation in mtr4 mutants. Taken together, these data link AtMTR4 with rRNA biogenesis and development in Arabidopsis. PMID:21682783

  14. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet

    PubMed Central

    Aiken, Catherine E.; Tarry-Adkins, Jane L.; Penfold, Naomi C.; Dearden, Laura; Ozanne, Susan E.

    2016-01-01

    Maternal diet during pregnancy influences the later life reproductive potential of female offspring. We investigate the molecular mechanisms underlying the depletion of ovarian follicular reserve in young adult females following exposure to obesogenic diet in early life. Furthermore, we explore the interaction between adverse maternal diet and postweaning diet in generating reduced ovarian reserve. Female mice were exposed to either maternal obesogenic (high fat/high sugar) or maternal control diet in utero and during lactation, then weaned onto either obesogenic or control diet. At 12 wk of age, the offspring ovarian reserve was depleted following exposure to maternal obesogenic diet (P < 0.05), but not postweaning obesogenic diet. Maternal obesogenic diet was associated with increased mitochondrial DNA biogenesis (copy number P < 0.05; transcription factor A, mitochondrial expression P < 0.05), increased mitochondrial antioxidant defenses [manganese superoxide dismutase (MnSOD) P < 0.05; copper/zinc superoxide dismutase P < 0.05; glutathione peroxidase 4 P < 0.01] and increased lipoxygenase expression (arachidonate 12-lipoxygenase P < 0.05; arachidonate 15-lipoxygenase P < 0.05) in the ovary. There was also significantly increased expression of the transcriptional regulator NF-κB (P < 0.05). There was no effect of postweaning diet on any measured ovarian parameters. Maternal diet thus plays a central role in determining follicular reserve in adult female offspring. Our observations suggest that lipid peroxidation and mitochondrial biogenesis are the key intracellular pathways involved in programming of ovarian reserve.—Aiken, C. E., Tarry-Adkins, J. L., Penfold, N. C., Dearden, L., Ozanne, S. E. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet. PMID:26700734

  15. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet.

    PubMed

    Aiken, Catherine E; Tarry-Adkins, Jane L; Penfold, Naomi C; Dearden, Laura; Ozanne, Susan E

    2016-04-01

    Maternal diet during pregnancy influences the later life reproductive potential of female offspring. We investigate the molecular mechanisms underlying the depletion of ovarian follicular reserve in young adult females following exposure to obesogenic diet in early life. Furthermore, we explore the interaction between adverse maternal diet and postweaning diet in generating reduced ovarian reserve. Female mice were exposed to either maternal obesogenic (high fat/high sugar) or maternal control dietin uteroand during lactation, then weaned onto either obesogenic or control diet. At 12 wk of age, the offspring ovarian reserve was depleted following exposure to maternal obesogenic diet (P< 0.05), but not postweaning obesogenic diet. Maternal obesogenic diet was associated with increased mitochondrial DNA biogenesis (copy numberP< 0.05; transcription factor A, mitochondrial expressionP< 0.05), increased mitochondrial antioxidant defenses [manganese superoxide dismutase (MnSOD)P< 0.05; copper/zinc superoxide dismutaseP< 0.05; glutathione peroxidase 4P< 0.01] and increased lipoxygenase expression (arachidonate 12-lipoxygenaseP< 0.05; arachidonate 15-lipoxygenaseP< 0.05) in the ovary. There was also significantly increased expression of the transcriptional regulator NF-κB (P< 0.05). There was no effect of postweaning diet on any measured ovarian parameters. Maternal diet thus plays a central role in determining follicular reserve in adult female offspring. Our observations suggest that lipid peroxidation and mitochondrial biogenesis are the key intracellular pathways involved in programming of ovarian reserve.-Aiken, C. E., Tarry-Adkins, J. L., Penfold, N. C., Dearden, L., Ozanne, S. E. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet. PMID:26700734

  16. A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.; Arimbasseri, Aneeshkumar G.

    2014-01-01

    Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to S. pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5′ and 3′ processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3′ oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3′ oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a ‘technical approaches’ section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses. PMID:25447915

  17. Ribosome biogenesis in skeletal development and the pathogenesis of skeletal disorders.

    PubMed

    Trainor, Paul A; Merrill, Amy E

    2014-06-01

    The skeleton affords a framework and structural support for vertebrates, while also facilitating movement, protecting vital organs, and providing a reservoir of minerals and cells for immune system and vascular homeostasis. The mechanical and biological functions of the skeleton are inextricably linked to the size and shape of individual bones, the diversity of which is dependent in part upon differential growth and proliferation. Perturbation of bone development, growth and proliferation, can result in congenital skeletal anomalies, which affect approximately 1 in 3000 live births [1]. Ribosome biogenesis is integral to all cell growth and proliferation through its roles in translating mRNAs and building proteins. Disruption of any steps in the process of ribosome biogenesis can lead to congenital disorders termed ribosomopathies. In this review, we discuss the role of ribosome biogenesis in skeletal development and in the pathogenesis of congenital skeletal anomalies. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. PMID:24252615

  18. Pharmacological induction of mitochondrial biogenesis as a therapeutic strategy for the treatment of type 2 diabetes.

    PubMed

    Zamora, Mònica; Pardo, Rosario; Villena, Josep A

    2015-11-01

    Defects in mitochondrial oxidative function have been associated with the onset of type 2 diabetes. Although the causal relationship between mitochondrial dysfunction and diabetes has not been fully established, numerous studies indicate that improved glucose homeostasis achieved via lifestyle interventions, such as exercise or calorie restriction, is tightly associated with increased mitochondrial biogenesis and oxidative function. Therefore, it is conceivable that potentiating mitochondrial biogenesis by pharmacological means could constitute an efficacious therapeutic strategy that would particularly benefit those diabetic patients who cannot adhere to comprehensive programs based on changes in lifestyle or that require a relatively rapid improvement in their diabetic status. In this review, we discuss several pharmacological targets and drugs that modulate mitochondrial biogenesis as well as their potential use as treatments for insulin resistance and diabetes. PMID:26212547

  19. Basal body structure and cell cycle-dependent biogenesis in Trypanosoma brucei.

    PubMed

    Vaughan, Sue; Gull, Keith

    2015-01-01

    Basal bodies are microtubule-based organelles that assemble cilia and flagella, which are critical for motility and sensory functions in all major eukaryotic lineages. The core structure of the basal body is highly conserved, but there is variability in biogenesis and additional functions that are organism and cell type specific. Work carried out in the protozoan parasite Trypanosoma brucei has arguably produced one of the most detailed dissections of basal body structure and biogenesis within the context of the flagellar pocket and associated organelles. In this review, we provide a detailed overview of the basic basal body structure in T. brucei along with the accessory structures and show how basal body movements during the basal body duplication cycle orchestrate cell and organelle morphogenesis. With this in-depth three-dimensional knowledge, identification of many basal body genes coupled with excellent genetic tools makes it an attractive model organism to study basal body biogenesis and maintenance. PMID:26862392

  20. T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases

    PubMed Central

    2011-01-01

    In skeletal muscle, the excitation-contraction (EC) coupling machinery mediates the translation of the action potential transmitted by the nerve into intracellular calcium release and muscle contraction. EC coupling requires a highly specialized membranous structure, the triad, composed of a central T-tubule surrounded by two terminal cisternae from the sarcoplasmic reticulum. While several proteins located on these structures have been identified, mechanisms governing T-tubule biogenesis and triad formation remain largely unknown. Here, we provide a description of triad structure and plasticity and review the role of proteins that have been linked to T-tubule biogenesis and triad formation and/or maintenance specifically in skeletal muscle: caveolin 3, amphiphysin 2, dysferlin, mitsugumins, junctophilins, myotubularin, ryanodine receptor, and dihydhropyridine Receptor. The importance of these proteins in triad biogenesis and subsequently in muscle contraction is sustained by studies on animal models and by the direct implication of most of these proteins in human myopathies. PMID:21797990

  1. Artemisinin mimics calorie restriction to trigger mitochondrial biogenesis and compromise telomere shortening in mice

    PubMed Central

    Wu, Ming; Li, Si-Ming; Gao, Qian

    2015-01-01

    Calorie restriction is known to extend lifespan among organisms by a debating mechanism underlying nitric oxide-driven mitochondrial biogenesis. We report here that nitric oxide generators including artemisinin, sodium nitroprusside, and L-arginine mimics calorie restriction and resembles hydrogen peroxide to initiate the nitric oxide signaling cascades and elicit the global antioxidative responses in mice. The large quantities of antioxidant enzymes are correlated with the low levels of reactive oxygen species, which allow the down-regulation of tumor suppressors and accessory DNA repair partners, eventually leading to the compromise of telomere shortening. Accompanying with the up-regulation of signal transducers and respiratory chain signatures, mitochondrial biogenesis occurs with the elevation of adenosine triphosphate levels upon exposure of mouse skeletal muscles to the mimetics of calorie restriction. In conclusion, calorie restriction-triggered nitric oxide provides antioxidative protection and alleviates telomere attrition via mitochondrial biogenesis, thereby maintaining chromosomal stability and integrity, which are the hallmarks of longevity. PMID:25780774

  2. Global effects of the small RNA biogenesis machinery on the Arabidopsis thaliana transcriptome.

    PubMed

    Laubinger, Sascha; Zeller, Georg; Henz, Stefan R; Buechel, Sabine; Sachsenberg, Timo; Wang, Jia-Wei; Rätsch, Gunnar; Weigel, Detlef

    2010-10-12

    In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. Our analysis revealed a surprising number of common targets in dcl1 and dcl2 dcl3 dcl4 triple mutants. Furthermore, our results suggest that the DCL1 is not only involved in miRNA action but also contributes to silencing of a subset of transposons, apparently through an effect on DNA methylation. PMID:20870966

  3. Biogenesis of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Holotoxin

    PubMed Central

    Ueno, Yoko; Ohara, Masaru; Kawamoto, Toru; Fujiwara, Tamaki; Komatsuzawa, Hitoshi; Oswald, Eric; Sugai, Motoyuki

    2006-01-01

    The cell cycle G2/M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA, cdtB, and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called “lipobox”, at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [3H]palmitate or [3H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA′), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA′ in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant. PMID:16714579

  4. Mitochondrial Retrograde Signaling: Triggers, Pathways, and Outcomes

    PubMed Central

    da Cunha, Fernanda Marques; Torelli, Nicole Quesada; Kowaltowski, Alicia J.

    2015-01-01

    Mitochondria are essential organelles for eukaryotic homeostasis. Although these organelles possess their own DNA, the vast majority (>99%) of mitochondrial proteins are encoded in the nucleus. This situation makes systems that allow the communication between mitochondria and the nucleus a requirement not only to coordinate mitochondrial protein synthesis during biogenesis but also to communicate eventual mitochondrial malfunctions, triggering compensatory responses in the nucleus. Mitochondria-to-nucleus retrograde signaling has been described in various organisms, albeit with differences in effector pathways, molecules, and outcomes, as discussed in this review. PMID:26583058

  5. N-acetylcysteine inhibits the up-regulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Chronic ethanol (EtOH) administration to experimental animals induces hepatic oxidative stress and up-regulates mitochondrial biogenesis. The mechanisms by which chronic EtOH up-regulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative ...

  6. Disorders of Lysosome-related Organelle Biogenesis: Clinical and Molecular Genetics

    PubMed Central

    Huizing, Marjan; Helip-Wooley, Amanda; Westbroek, Wendy; Gunay-Aygun, Meral; Gahl, William A.

    2009-01-01

    Lysosome-related organelles (LROs) are a heterogeneous group of vesicles that share various features with lysosomes, but are distinct in function, morphology, and composition. The biogenesis of LROs employs a common machinery, and genetic defects in this machinery can affect all LROs or only an individual LRO, resulting in a variety of clinical features. In this review, we discuss the main components in LRO biogenesis. We also address the function, composition and resident cell type of the major LROs. Finally, we describe the clinical characteristics of the major human LRO disorders. PMID:18544035

  7. Assay Methods for H2S Biogenesis and Catabolism Enzymes

    PubMed Central

    Banerjee, Ruma; Chiku, Taurai; Kabil, Omer; Libiad, Marouane; Motl, Nicole; Yadav, Pramod K.

    2015-01-01

    H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine β-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter. PMID:25725523

  8. Assay methods for H2S biogenesis and catabolism enzymes.

    PubMed

    Banerjee, Ruma; Chiku, Taurai; Kabil, Omer; Libiad, Marouane; Motl, Nicole; Yadav, Pramod K

    2015-01-01

    H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine β-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter. PMID:25725523

  9. Repression of microRNA biogenesis by silencing of OsDCL1 activates the basal resistance to Magnaporthe oryzae in rice.

    PubMed

    Zhang, Dandan; Liu, Muxing; Tang, Mingzhi; Dong, Bo; Wu, Dianxing; Zhang, Zhengguang; Zhou, Bo

    2015-08-01

    The RNaseIII enzyme Dicer-like 1 (DCL1) processes the microRNA biogenesis and plays a determinant role in plant development. In this study, we reported the function of OsDCL1 in the immunity to rice blast, the devastating disease caused by the fungal pathogen, Magnaporthe oryzae. Expression profiling demonstrated that different OsDCLs responded dynamically and OsDCL1 reduced its expression upon the challenge of rice blast pathogen. In contrast, miR162a predicted to target OsDCL1 increased its expression, implying a negative feedback loop between OsDCL1 and miR162a in rice. In addition to developmental defects, the OsDCL1-silencing mutants showed enhanced resistance to virulent rice blast strains in a non-race specific manner. Accumulation of hydrogen peroxide and cell death were observed in the contact cells with infectious hyphae, revealing that silencing of OsDCL1 activated cellular defense responses. In OsDCL1 RNAi lines, 12 differentially expressed miRNAs were identified, of which 5 and 7 were down- and up-regulated, respectively, indicating that miRNAs responded dynamically in the interaction between rice and rice blast. Moreover, silencing of OsDCL1 activated the constitutive expression of defense related genes. Taken together, our results indicate that rice is capable of activating basal resistance against rice blast by perturbing OsDCL1-dependent miRNA biogenesis pathway. PMID:26089149

  10. Drosophila Mbm Is a Nucleolar Myc and Casein Kinase 2 Target Required for Ribosome Biogenesis and Cell Growth of Central Brain Neuroblasts

    PubMed Central

    Hovhanyan, Anna; Herter, Eva K.; Pfannstiel, Jens; Gallant, Peter

    2014-01-01

    Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy-sensing organs into growth-promoting processes, such as sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila melanogaster. Yet the molecular function of Mbm has so far been unknown. Here we show that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which, among other functions, relays information from nutrient-dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by casein kinase 2 (CK2), which has an impact on localization of the protein. We conclude that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which, together with CK2-mediated signals, enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth. PMID:24615015

  11. Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes.

    PubMed

    Zambetti, Noemi A; Bindels, Eric M J; Van Strien, Paulina M H; Valkhof, Marijke G; Adisty, Maria N; Hoogenboezem, Remco M; Sanders, Mathijs A; Rommens, Johanna M; Touw, Ivo P; Raaijmakers, Marc H G P

    2015-10-01

    Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome. PMID:26185170

  12. Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes

    PubMed Central

    Zambetti, Noemi A.; Bindels, Eric M. J.; Van Strien, Paulina M. H.; Valkhof, Marijke G.; Adisty, Maria N.; Hoogenboezem, Remco M.; Sanders, Mathijs A.; Rommens, Johanna M.; Touw, Ivo P.; Raaijmakers, Marc H. G. P.

    2015-01-01

    Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome. PMID:26185170

  13. Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis

    PubMed Central

    Moriggi, Giulia; Nieto, Blanca; Dosil, Mercedes

    2014-01-01

    During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes. PMID:25474739

  14. Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe–S cluster biogenesis regulation

    PubMed Central

    Ciesielski, Szymon J.; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A.

    2016-01-01

    Iron–sulfur (Fe–S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe–S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe–S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe–S cluster biogenesis does not meet cellular demands. PMID:26842892

  15. Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

    PubMed

    Ciesielski, Szymon J; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A

    2016-04-01

    Iron-sulfur (Fe-S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands. PMID:26842892

  16. Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

    PubMed Central

    Jung, Su Ryun; Asaka, Meiko; Holloszy, John O.; Han, Dong-Ho

    2013-01-01

    It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C2C12 myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle. PMID:23874150

  17. Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

    PubMed Central

    Bohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.; von der Malsburg, Karina; Müller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid, Bettina; Chacinska, Agnieszka; Veenhuis, Marten; van der Klei, Ida J.; Daum, Günther; Wiedemann, Nils; Becker, Thomas; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins. PMID:22918945

  18. Expression of Flotilin-2 and Acrosome Biogenesis Are Regulated by MiR-124 during Spermatogenesis

    PubMed Central

    Zheng, Haoyu; Jiang, Min; Xia, Zhengrong; Yu, Jinjin; Chen, Ling; Huang, Xiaoyan

    2015-01-01

    MicroRNAs (miRNAs) are a class of short non-coding RNA molecules, which diversely regulate gene expression in organisms. Although the regulatory role of these small RNA molecules has been recently explored in animal spermatogenesis, the role of miR-124 in male germ cells is poorly defined. In our previous study, flotillin-2 was investigated as a novel Golgi-related protein involved in sperm acrosome biogenesis. The current study was designed to analyze the contribution of miR-124 in the regulation of flotillin-2 expression during mouse acrosome biogenesis. Luciferase assays revealed the target effects of miR-124 on flotillin-2 expression. Following intratesticular injection of miR-124 in 3-week-old male mice, quantitative real-time RT-PCR and western blot analysis were employed to confirm the function of miR-124 in regulating flotillin-2 after 48 hours. Sperm abnormalities were assessed 3 weeks later by ordinary optical microscopy, the acrosome abnormalities were also assessed by PNA staining and transmission electron microscopy. The results showed the proportion of sperm acrosome abnormalities was significantly higher than that of the control group. The expression of flotillin-2 and caveolin-1 was significantly downregulated during acrosome biogenesis. These results indicated that miR-124 could potentially play a role in caveolin-independent vesicle trafficking and modulation of flotillin-2 expression in mouse acrosome biogenesis. PMID:26313572

  19. Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells.

    PubMed

    Marine, Akira; Krager, Kimberly J; Aykin-Burns, Nukhet; Macmillan-Crow, Lee Ann

    2014-01-01

    Superoxide is widely regarded as the primary reactive oxygen species (ROS) which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK) cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ), a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS) inhibitor demonstrated that peroxynitrite (at low micromolar levels) induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation. PMID:24563852

  20. Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: Modulation by antioxidants

    SciTech Connect

    Perez-de-Arce, Karen; Foncea, Rocio . E-mail: rfoncea@med.puc.cl; Leighton, Federico

    2005-12-16

    It has been proposed that homocysteine (Hcy)-induces endothelial dysfunction and atherosclerosis by generation of reactive oxygen species (ROS). A previous report has shown that Hcy promotes mitochondrial damage. Considering that oxidative stress can affect mitochondrial biogenesis, we hypothesized that Hcy-induced ROS in endothelial cells may lead to increased mitochondrial biogenesis. We found that Hcy-induced ROS (1.85-fold), leading to a NF-{kappa}B activation and increase the formation of 3-nitrotyrosine. Furthermore, expression of the mitochondrial biogenesis factors, nuclear respiratory factor-1 and mitochondrial transcription factor A, was significantly elevated in Hcy-treated cells. These changes were accompanied by increase in mitochondrial mass and higher mRNA and protein expression of the subunit III of cytochrome c oxidase. These effects were significantly prevented by pretreatment with the antioxidants, catechin and trolox. Taken together, our results suggest that ROS is an important mediator of mitochondrial biogenesis induced by Hcy, and that modulation of oxidative stress by antioxidants may protect against the adverse vascular effects of Hcy.

  1. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  2. Proteome distribution between nucleoplasm and nucleolus and its relation to ribosome biogenesis in Arabidopsis thaliana.

    PubMed

    Palm, Denise; Simm, Stefan; Darm, Katrin; Weis, Benjamin L; Ruprecht, Maike; Schleiff, Enrico; Scharf, Christian

    2016-01-01

    Ribosome biogenesis is an essential process initiated in the nucleolus. In eukaryotes, multiple ribosome biogenesis factors (RBFs) can be found in the nucleolus, the nucleus and in the cytoplasm. They act in processing, folding and modification of the pre-ribosomal (r)RNAs, incorporation of ribosomal proteins (RPs), export of pre-ribosomal particles to the cytoplasm, and quality control mechanisms. Ribosome biogenesis is best established for Saccharomyces cerevisiae. Plant ortholog assignment to yeast RBFs revealed the absence of about 30% of the yeast RBFs in plants. In turn, few plant specific proteins have been identified by biochemical experiments to act in plant ribosome biogenesis. Nevertheless, a complete inventory of plant RBFs has not been established yet. We analyzed the proteome of the nucleus and nucleolus of Arabidopsis thaliana and the post-translational modifications of these proteins. We identified 1602 proteins in the nucleolar and 2544 proteins in the nuclear fraction with an overlap of 1429 proteins. For a randomly selected set of proteins identified by the proteomic approach we confirmed the localization inferred from the proteomics data by the localization of GFP fusion proteins. We assigned the identified proteins to various complexes and functions and found about 519 plant proteins that have a potential to act as a RBFs, but which have not been experimentally characterized yet. Last, we compared the distribution of RBFs and RPs in the various fractions with the distribution established for yeast. PMID:26980300

  3. Atg7 is required for acrosome biogenesis during spermatogenesis in mice

    PubMed Central

    Wang, Hongna; Wan, Haifeng; Li, Xixia; Liu, Weixiao; Chen, Qi; Wang, Yaqing; Yang, Lin; Tang, Hongmei; Zhang, Xiujun; Duan, Enkui; Zhao, Xiaoyang; Gao, Fei; Li, Wei

    2014-01-01

    The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in the process of fertilization. The molecular mechanism underlying the biogenesis of this lysosome-related organelle (LRO) is still largely unknown. Here, we show that germ cell-specific Atg7-knockout mice were infertile due to a defect in acrosome biogenesis and displayed a phenotype similar to human globozoospermia; this reproductive defect was successfully rescued by intracytoplasmic sperm injections. Furthermore, the depletion of Atg7 in germ cells did not affect the early stages of development of germ cells, but at later stages of spermatogenesis, the proacrosomal vesicles failed to fuse into a single acrosomal vesicle during the Golgi phase, which finally resulted in irregular or nearly round-headed spermatozoa. Autophagic flux was disrupted in Atg7-depleted germ cells, finally leading to the failure of LC3 conjugation to Golgi apparatus-derived vesicles. In addition, Atg7 partially regulated another globozoospermia-related protein, Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), during acrosome biogenesis. Finally, the injection of either autophagy or lysosome inhibitors into testis resulted in a similar phenotype to that of germ cell-specific Atg7-knockout mice. Altogether, our results uncover a new role for Atg7 in the biogenesis of the acrosome, and we provide evidence to support the autolysosome origination hypothesis for the acrosome. PMID:24853953

  4. Acyl-CoA N-acyltransferase influences fertility by regulating lipid metabolism and jasmonic acid biogenesis in cotton

    PubMed Central

    Fu, Wenfeng; Shen, Ying; Hao, Juan; Wu, Jianyong; Ke, Liping; Wu, Caiyun; Huang, Kai; Luo, Binglun; Xu, Mingfeng; Cheng, Xiaofei; Zhou, Xueping; Sun, Jie; Xing, Chaozhu; Sun, Yuqiang

    2015-01-01

    Cotton (Gossypium spp.) is an important economic crop and there is obvious heterosis in cotton, fertility has played an important role in this heterosis. However, the genes that exhibit critical roles in anther development and fertility are not well understood. Here, we report an acyl-CoA N-acyltransferase (EC2.3; GhACNAT) that plays a key role in anther development and fertility. Suppression of GhACNAT by virus-induced gene silencing in transgenic cotton (G. hirsutum L. cv. C312) resulted in indehiscent anthers that were full of pollen, diminished filaments and stamens, and plant sterility. We found GhACNAT was involved in lipid metabolism and jasmonic acid (JA) biosynthesis. The genes differentially expressed in GhACNAT-silenced plants and C312 were mainly involved in catalytic activity and transcription regulator activity in lipid metabolism. In GhACNAT-silenced plants, the expression levels of genes involved in lipid metabolism and jasmonic acid biosynthesis were significantly changed, the amount of JA in leaves and reproductive organs was significantly decreased compared with the amounts in C312. Treatments with exogenous methyl jasmonate rescued anther dehiscence and pollen release in GhACNAT-silenced plants and caused self-fertility. The GhACNAT gene may play an important role in controlling cotton fertility by regulating the pathways of lipid synthesis and JA biogenesis. PMID:26134787

  5. Novel structural co-expression analysis linking the NPM1-associated ribosomal biogenesis network to chronic myelogenous leukemia

    PubMed Central

    Chan, Lawrence WC; Lin, Xihong; Yung, Godwin; Lui, Thomas; Chiu, Ya Ming; Wang, Fengfeng; Tsui, Nancy BY; Cho, William CS; Yip, SP; Siu, Parco M.; Wong, SC Cesar; Yung, Benjamin YM

    2015-01-01

    Co-expression analysis reveals useful dysregulation patterns of gene cooperativeness for understanding cancer biology and identifying new targets for treatment. We developed a structural strategy to identify co-expressed gene networks that are important for chronic myelogenous leukemia (CML). This strategy compared the distributions of expressional correlations between CML and normal states, and it identified a data-driven threshold to classify strongly co-expressed networks that had the best coherence with CML. Using this strategy, we found a transcriptome-wide reduction of co-expression connectivity in CML, reflecting potentially loosened molecular regulation. Conversely, when we focused on nucleophosmin 1 (NPM1) associated networks, NPM1 established more co-expression linkages with BCR-ABL pathways and ribosomal protein networks in CML than normal. This finding implicates a new role of NPM1 in conveying tumorigenic signals from the BCR-ABL oncoprotein to ribosome biogenesis, affecting cellular growth. Transcription factors may be regulators of the differential co-expression patterns between CML and normal. PMID:26205693

  6. Short-Chain Fatty Acid Acetate Stimulates Adipogenesis and Mitochondrial Biogenesis via GPR43 in Brown Adipocytes.

    PubMed

    Hu, Jiamiao; Kyrou, Ioannis; Tan, Bee K; Dimitriadis, Georgios K; Ramanjaneya, Manjunath; Tripathi, Gyanendra; Patel, Vanlata; James, Sean; Kawan, Mohamed; Chen, Jing; Randeva, Harpal S

    2016-05-01

    Short-chain fatty acids play crucial roles in a range of physiological functions. However, the effects of short-chain fatty acids on brown adipose tissue have not been fully investigated. We examined the role of acetate, a short-chain fatty acid formed by fermentation in the gut, in the regulation of brown adipocyte metabolism. Our results show that acetate up-regulates adipocyte protein 2, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 expression and affects the morphological changes of brown adipocytes during adipogenesis. Moreover, an increase in mitochondrial biogenesis was observed after acetate treatment. Acetate also elicited the activation of ERK and cAMP response element-binding protein, and these responses were sensitive to G(i/o)-type G protein inactivator, Gβγ-subunit inhibitor, phospholipase C inhibitor, and MAPK kinase inhibitor, indicating a role for the G(i/o)βγ/phospholipase C/protein kinase C/MAPK kinase signaling pathway in these responses. These effects of acetate were mimicked by treatment with 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide, a synthetic G protein-coupled receptor 43 (GPR43) agonist and were impaired in GPR43 knockdown cells. Taken together, our results indicate that acetate may have important physiological roles in brown adipocytes through the activation of GPR43. PMID:26990063

  7. Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

    PubMed

    Gurunathan, Sangiliyandi; David, Doris; Gerst, Jeffrey E

    2002-02-15

    Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis. PMID:11847108

  8. Stimulatory Effects of Balanced Deep Sea Water on Mitochondrial Biogenesis and Function.

    PubMed

    Ha, Byung Geun; Park, Jung-Eun; Cho, Hyun-Jung; Shon, Yun Hee

    2015-01-01

    The worldwide prevalence of metabolic diseases, including obesity and diabetes, is increasing. Mitochondrial dysfunction is recognized as a core feature of these diseases. Emerging evidence also suggests that defects in mitochondrial biogenesis, number, morphology, fusion, and fission, contribute to the development and progression of metabolic diseases. Our previous studies revealed that balanced deep-sea water (BDSW) has potential as a treatment for diabetes and obesity. In this study, we aimed to investigate the mechanism by which BDSW regulates diabetes and obesity by studying its effects on mitochondrial metabolism. To determine whether BDSW regulates mitochondrial biogenesis and function, we investigated its effects on mitochondrial DNA (mtDNA) content, mitochondrial enzyme activity, and the expression of transcription factors and mitochondria specific genes, as well as on the phosphorylation of signaling molecules associated with mitochondria biogenesis and its function in C2C12 myotubes. BDSW increased mitochondrial biogenesis in a time and dose-dependent manner. Quantitative real-time PCR revealed that BDSW enhances gene expression of PGC-1α, NRF1, and TFAM for mitochondrial transcription; MFN1/2 and DRP1 for mitochondrial fusion; OPA1 for mitochondrial fission; TOMM40 and TIMM44 for mitochondrial protein import; CPT-1α and MCAD for fatty acid oxidation; CYTC for oxidative phosphorylation. Upregulation of these genes was validated by increased mitochondria staining, CS activity, CytC oxidase activity, NAD+ to NADH ratio, and the phosphorylation of signaling molecules such as AMPK and SIRT1. Moreover, drinking BDSW remarkably improved mtDNA content in the muscles of HFD-induced obese mice. Taken together, these results suggest that the stimulatory effect of BDSW on mitochondrial biogenesis and function may provide further insights into the regulatory mechanism of BDSW-induced anti-diabetic and anti-obesity action. PMID:26068191

  9. N-acetylcysteine inhibits the upregulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    PubMed Central

    Caro, Andres A.; Bell, Matthew; Ejiofor, Shannon; Zurcher, Grant; Petersen, Dennis R.; Ronis, Martin J. J.

    2014-01-01

    Background Chronic ethanol administration to experimental animals induces hepatic oxidative stress and upregulates mitochondrial biogenesis. The mechanisms by which chronic ethanol upregulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic ethanol feeding. If our hypothesis is correct, co-administration of antioxidants should prevent upregulation of mitochondrial biogenesis genes. Methods Rats were fed an ethanol-containing diet intragastrically by total enteral nutrition for 150 days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7 g/kg/day; control rats were administered isocaloric diets where carbohydrates substituted for ethanol calories. Results Ethanol administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and GSH/GSSG ratio. These effects were inhibited by co-administration of ethanol and NAC. Chronic ethanol increased the expression of mitochondrial biogenesis genes including peroxisome proliferator activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of ethanol and NAC prevented these effects. Chronic ethanol administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC. Conclusions These results suggest that oxidative stress caused by chronic ethanol triggered the upregulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by

  10. Overproduction of CcmG and CcmFHRc Fully Suppresses the c-Type Cytochrome Biogenesis Defect of Rhodobacter capsulatus CcmI-Null Mutants

    PubMed Central

    Sanders, Carsten; Deshmukh, Meenal; Astor, Doniel; Kranz, Robert G.; Daldal, Fevzi

    2005-01-01

    Gram-negative bacteria like Rhodobacter capsulatus use intertwined pathways to carry out the posttranslational maturation of c-type cytochromes (Cyts). This periplasmic process requires at least 10 essential components for apo-Cyt c chaperoning, thio-oxidoreduction, and the delivery of heme and its covalent ligation. One of these components, CcmI (also called CycH), is thought to act as an apo-Cyt c chaperone. In R. capsulatus, CcmI-null mutants are unable to produce c-type Cyts and thus sustain photosynthetic (Ps) growth. Previously, we have shown that overproduction of the putative heme ligation components CcmF and CcmHRc (also called Ccl1 and Ccl2) can partially bypass the function of CcmI on minimal, but not on enriched, media. Here, we demonstrate that either additional overproduction of CcmG (also called HelX) or hyperproduction of CcmF-CcmHRc is needed to completely overcome the role of CcmI during the biogenesis of c-type Cyts on both minimal and enriched media. These findings indicate that, in the absence of CcmI, interactions between the heme ligation and thioreduction pathways become restricted for sufficient Cyt c production. We therefore suggest that CcmI, along with its apo-Cyt chaperoning function, is also critical for the efficacy of holo-Cyt c formation, possibly via its close interactions with other components performing the final heme ligation steps during Cyt c biogenesis. PMID:15937187

  11. Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

    PubMed

    Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

    2008-01-01

    The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex. PMID:17974559

  12. Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscle by hypoxia preconditioning with cobalt chloride

    SciTech Connect

    Saxena, Saurabh; Shukla, Dhananjay; Bansal, Anju

    2012-11-01

    High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hypoxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle to increase physical performance. Cobalt chloride (CoCl{sub 2}), a hypoxia mimetic, stabilizes HIF-1, which otherwise is degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl{sub 2} supplementation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. The results showed significant increase in physical performance in cobalt supplemented rats without (two times) or with training (3.3 times) as compared to control animals. CoCl{sub 2} supplementation in rats augmented the biological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased the expression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobic respiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increased mRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microscopy. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seems to be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) induction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl{sub 2} supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic respiration in skeletal muscle, which leads to increased physical performance. The significance of this study lies in understanding the molecular mechanism of hypoxia adaptation and improvement of work performance in normal as well as extreme conditions like hypoxia via hypoxia preconditioning. -- Highlights: ► We supplemented rats with CoCl{sub 2} for 15 days along with training. ► Co

  13. Studying lipids involved in the endosomal pathway.

    PubMed

    Bissig, Christin; Johnson, Shem; Gruenberg, Jean

    2012-01-01

    Endosomes along the degradation pathway exhibit a multivesicular appearance and differ in their lipid compositions. Association of proteins to specific membrane lipids and presumably also lipid-lipid interactions contribute to the formation of functional membrane platforms that regulate endosome biogenesis and function. This chapter provides a brief review of the functions of endosomal lipids in the degradation pathway, a discussion of techniques that allow studying lipid-based mechanisms and a selection of step-by-step protocols for in vivo and in vitro methods commonly used to study lipid roles in endocytosis. The techniques described here have been used to elucidate the function of the late endosomal lipid lysobisphosphatidic acid and allow the monitoring of lipid distribution, levels and dynamics, as well as the characterization of lipid-binding partners. PMID:22325596

  14. The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries

    PubMed Central

    Nigg, Erich A.; Stearns, Tim

    2013-01-01

    Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes. PMID:21968988

  15. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis

    PubMed Central

    Jeschke, Andreas; Zehethofer, Nicole; Lindner, Buko; Krupp, Jessica; Schwudke, Dominik; Haneburger, Ina; Jovic, Marko; Backer, Jonathan M.; Balla, Tamas; Hilbi, Hubert; Haas, Albert

    2015-01-01

    Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway. PMID:25825728

  16. VAMP7 Modulates Ciliary Biogenesis in Kidney Cells

    PubMed Central

    Szalinski, Christina M.; Labilloy, Anatália; Bruns, Jennifer R.; Weisz, Ora A.

    2014-01-01

    Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. PMID:24466086

  17. miRNA biogenesis: biological impact in the development of cancer.

    PubMed

    Romero-Cordoba, Sandra L; Salido-Guadarrama, Ivan; Rodriguez-Dorantes, Mauricio; Hidalgo-Miranda, Alfredo

    2014-01-01

    microRNAs (miRNAs) are non coding RNAs with different biological functions and pathological implications. Given their role as post-transcriptional gene expression regulators, they are involved in several important physiological processes like development, cell differentiation and cell signaling. miRNAs act as modulators of gene expression programs in different diseases, particularly in cancer, where they act through the repression of genes which are critical for carcinogenesis. The expression level of mature miRNAs is the result of a fine mechanism of biogenesis, carried out by different enzymatic complexes that exert their function at transcriptional and post-transcriptional levels. In this review, we will focus our discussion on the alterations in the miRNA biogenesis machinery, and its impact on the establishment and development of cancer programs. PMID:25482951

  18. Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1

    PubMed Central

    Yao, Andrew I.; Facciotti, Marc T.

    2011-01-01

    It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600). In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis. PMID:22110395

  19. Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels

    PubMed Central

    Coessens, Elise; Manifava, Maria; Georgiev, Plamen; Pettitt, Trevor; Wood, Eleanor; Garcia-Murillas, Isaac; Okkenhaug, Hanneke; Trivedi, Deepti; Zhang, Qifeng; Razzaq, Azam; Zaid, Ola; Wakelam, Michael; O'Kane, Cahir J; Ktistakis, Nicholas

    2009-01-01

    Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis. PMID:19349583

  20. piRNA-guided slicing specifies transcripts for Zucchini dependent, phased piRNA biogenesis

    PubMed Central

    Brennecke, Julius

    2016-01-01

    In animal gonads PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound piRNAs. These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3′ directed and phased process that, in the Drosophila germline, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endo-nucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3′ and 5′ ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3′ end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism where Zucchini plays a central role in defining piRNA 5′ and 3′ ends. PMID:25977553

  1. Tetramethylpyrazine ameliorates high glucose-induced endothelial dysfunction by increasing mitochondrial biogenesis.

    PubMed

    Xu, Qiong; Xia, Pu; Li, Xi; Wang, Wei; Liu, Zhenqi; Gao, Xin

    2014-01-01

    Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of inflammatory and cardiovascular diseases. We have previously reported that TMP acts as a potent antioxidant protecting endothelial cells against high glucose-induced damages. However, the molecular mechanism responsible for the antioxidant effect of TMP remains to be elucidated. In this study, we show that TMP increases nitric oxide production in endothelial cells and promotes endothelium-dependent relaxation in rate aortic rings. The antioxidant effect of TMP appears attributable to its ability to activate the mitochondrial biogenesis, as reflected in an up-regulation of complex III and amelioration of mitochondrial membrane potential. Furthermore, TMP is able to reverse high glucose-induced suppression of SIRT1 and the biogenesis-related factors, including PGC-1α, NRF1 and TFAM, suggesting a new molecular mechanism underlying the protective effect of TMP on the endothelium. PMID:24505445

  2. Tetramethylpyrazine Ameliorates High Glucose-Induced Endothelial Dysfunction by Increasing Mitochondrial Biogenesis

    PubMed Central

    Xu, Qiong; Xia, Pu; Li, Xi; Wang, Wei; Liu, Zhenqi; Gao, Xin

    2014-01-01

    Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of inflammatory and cardiovascular diseases. We have previously reported that TMP acts as a potent antioxidant protecting endothelial cells against high glucose-induced damages. However, the molecular mechanism responsible for the antioxidant effect of TMP remains to be elucidated. In this study, we show that TMP increases nitric oxide production in endothelial cells and promotes endothelium-dependent relaxation in rate aortic rings. The antioxidant effect of TMP appears attributable to its ability to activate the mitochondrial biogenesis, as reflected in an up-regulation of complex III and amelioration of mitochondrial membrane potential. Furthermore, TMP is able to reverse high glucose-induced suppression of SIRT1 and the biogenesis-related factors, including PGC-1α, NRF1 and TFAM, suggesting a new molecular mechanism underlying the protective effect of TMP on the endothelium. PMID:24505445

  3. A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila

    PubMed Central

    Muerdter, Felix; Guzzardo, Paloma M.; Gillis, Jesse; Luo, Yicheng; Yu, Yang; Chen, Caifu; Fekete, Richard; Hannon, Gregory J.

    2013-01-01

    Summary A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels. PMID:23665228

  4. PBR1 selectively controls biogenesis of photosynthetic complexes by modulating translation of the large chloroplast gene Ycf1 in Arabidopsis

    PubMed Central

    Yang, Xiao-Fei; Wang, Yu-Ting; Chen, Si-Ting; Li, Ji-Kai; Shen, Hong-Tao; Guo, Fang-Qing

    2016-01-01

    The biogenesis of photosystem I (PSI), cytochrome b6f (Cytb6f) and NADH dehydrogenase (NDH) complexes relies on the spatially and temporally coordinated expression and translation of both nuclear and chloroplast genes. Here we report the identification of photosystem biogenesis regulator 1 (PBR1), a nuclear-encoded chloroplast RNA-binding protein that regulates the concerted biogenesis of NDH, PSI and Cytb6f complexes. We identified Ycf1, one of the two largest chloroplast genome-encoded open reading frames as the direct downstream target protein of PBR1. Biochemical and molecular analyses reveal that PBR1 regulates Ycf1 translation by directly binding to its mRNA. Surprisingly, we further demonstrate that relocation of the chloroplast gene Ycf1 fused with a plastid-transit sequence to the nucleus bypasses the requirement of PBR1 for Ycf1 translation, which sufficiently complements the defects in biogenesis of NDH, PSI and Cytb6f complexes in PBR1-deficient plants. Remarkably, the nuclear-encoded PBR1 tightly controls the expression of the chloroplast gene Ycf1 at the translational level, which is sufficient to sustain the coordinated biogenesis of NDH, PSI and Cytb6f complexes as a whole. Our findings provide deep insights into better understanding of how a predominant nuclear-encoded factor can act as a migratory mediator and undergoes selective translational regulation of the target plastid gene in controlling biogenesis of photosynthetic complexes. PMID:27462450

  5. PBR1 selectively controls biogenesis of photosynthetic complexes by modulating translation of the large chloroplast gene Ycf1 in Arabidopsis.

    PubMed

    Yang, Xiao-Fei; Wang, Yu-Ting; Chen, Si-Ting; Li, Ji-Kai; Shen, Hong-Tao; Guo, Fang-Qing

    2016-01-01

    The biogenesis of photosystem I (PSI), cytochrome b 6 f (Cytb 6 f) and NADH dehydrogenase (NDH) complexes relies on the spatially and temporally coordinated expression and translation of both nuclear and chloroplast genes. Here we report the identification of photosystem biogenesis regulator 1 (PBR1), a nuclear-encoded chloroplast RNA-binding protein that regulates the concerted biogenesis of NDH, PSI and Cytb 6 f complexes. We identified Ycf1, one of the two largest chloroplast genome-encoded open reading frames as the direct downstream target protein of PBR1. Biochemical and molecular analyses reveal that PBR1 regulates Ycf1 translation by directly binding to its mRNA. Surprisingly, we further demonstrate that relocation of the chloroplast gene Ycf1 fused with a plastid-transit sequence to the nucleus bypasses the requirement of PBR1 for Ycf1 translation, which sufficiently complements the defects in biogenesis of NDH, PSI and Cytb 6 f complexes in PBR1-deficient plants. Remarkably, the nuclear-encoded PBR1 tightly controls the expression of the chloroplast gene Ycf1 at the translational level, which is sufficient to sustain the coordinated biogenesis of NDH, PSI and Cytb 6 f complexes as a whole. Our findings provide deep insights into better understanding of how a predominant nuclear-encoded factor can act as a migratory mediator and undergoes selective translational regulation of the target plastid gene in controlling biogenesis of photosynthetic complexes. PMID:27462450

  6. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

    SciTech Connect

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.

  7. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis

    NASA Astrophysics Data System (ADS)

    Finley, Daniel; Bartel, Bonnie; Varshavsky, Alexander

    1989-03-01

    Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.

  8. Yeast Mitochondria as a Model System to Study the Biogenesis of Bacterial β-Barrel Proteins.

    PubMed

    Ulrich, Thomas; Oberhettinger, Philipp; Autenrieth, Ingo B; Rapaport, Doron

    2015-01-01

    Beta-barrel proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The evolutionary conservation in the biogenesis of these proteins allows mitochondria to assemble bacterial β-barrel proteins in their functional form. In this chapter, we describe exemplarily how the capacity of yeast mitochondria to process the trimeric autotransporter YadA can be used to study the role of bacterial periplasmic chaperones in this process. PMID:26427673

  9. Nom1 Mediates Pancreas Development by Regulating Ribosome Biogenesis in Zebrafish

    PubMed Central

    Qin, Wei; Chen, Zelin; Zhang, Yihan; Yan, Ruibin; Yan, Guanrong; Li, Song; Zhong, Hanbing; Lin, Shuo

    2014-01-01

    Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1α into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation. PMID:24967912

  10. Role of dimerization in dopamine D(4) receptor biogenesis.

    PubMed

    Van Craenenbroeck, Kathleen; Borroto-Escuela, Dasiel O; Skieterska, Kamila; Duchou, Jolien; Romero-Fernandez, Wilber; Fuxe, Kjell

    2014-01-01

    Dopamine receptors are G protein-coupled receptors critically involved in locomotion, reward, and cognitive processes. Export of dopamine receptors to the plasma membrane is thought to follow the default secretory pathway, whereby proteins travel from the endoplasmatic reticulum (ER), through the Golgi apparatus, to arrive at the cell surface. Several observations indicate that trafficking from the ER to the plasma membrane is tightly regulated, and that correct folding in the ER acts as a bottle neck to the maturation of the dopamine D4 receptors. The dopamine D(4) receptor is an interesting receptor since it has a polymorphic region in its third intracellular loop, resulting in receptor isoforms of varying length and amino acid composition. Correct folding is enhanced by: (1) interaction with specific proteins, such as ER resident chaperones, (2) interaction with pharmacological chaperones, for example, ligands that are membrane permeable and can bind to the receptor in the ER, and (3) receptor dimerization; the assembly of multisubunit proteins into a quaternary structure is started in the ER before cell surface delivery, which helps in correct folding and subsequent expression. These interactions help the process of GPCR folding, but more importantly they ensure that only properly folded proteins proceed from the ER to the trans-Golgi network. In this review we will mainly focus on the role of receptor dimerization in dopamine D(4) receptor maturation. PMID:25175456

  11. Protein biogenesis machinery is a driver of replicative aging in yeast

    PubMed Central

    Janssens, Georges E; Meinema, Anne C; González, Javier; Wolters, Justina C; Schmidt, Alexander; Guryev, Victor; Bischoff, Rainer; Wit, Ernst C; Veenhoff, Liesbeth M; Heinemann, Matthias

    2015-01-01

    An integrated account of the molecular changes occurring during the process of cellular aging is crucial towards understanding the underlying mechanisms. Here, using novel culturing and computational methods as well as latest analytical techniques, we mapped the proteome and transcriptome during the replicative lifespan of budding yeast. With age, we found primarily proteins involved in protein biogenesis to increase relative to their transcript levels. Exploiting the dynamic nature of our data, we reconstructed high-level directional networks, where we found the same protein biogenesis-related genes to have the strongest ability to predict the behavior of other genes in the system. We identified metabolic shifts and the loss of stoichiometry in protein complexes as being consequences of aging. We propose a model whereby the uncoupling of protein levels of biogenesis-related genes from their transcript levels is causal for the changes occurring in aging yeast. Our model explains why targeting protein synthesis, or repairing the downstream consequences, can serve as interventions in aging. DOI: http://dx.doi.org/10.7554/eLife.08527.001 PMID:26422514

  12. Protein biogenesis machinery is a driver of replicative aging in yeast.

    PubMed

    Janssens, Georges E; Meinema, Anne C; González, Javier; Wolters, Justina C; Schmidt, Alexander; Guryev, Victor; Bischoff, Rainer; Wit, Ernst C; Veenhoff, Liesbeth M; Heinemann, Matthias

    2015-01-01

    An integrated account of the molecular changes occurring during the process of cellular aging is crucial towards understanding the underlying mechanisms. Here, using novel culturing and computational methods as well as latest analytical techniques, we mapped the proteome and transcriptome during the replicative lifespan of budding yeast. With age, we found primarily proteins involved in protein biogenesis to increase relative to their transcript levels. Exploiting the dynamic nature of our data, we reconstructed high-level directional networks, where we found the same protein biogenesis-related genes to have the strongest ability to predict the behavior of other genes in the system. We identified metabolic shifts and the loss of stoichiometry in protein complexes as being consequences of aging. We propose a model whereby the uncoupling of protein levels of biogenesis-related genes from their transcript levels is causal for the changes occurring in aging yeast. Our model explains why targeting protein synthesis, or repairing the downstream consequences, can serve as interventions in aging. PMID:26422514

  13. Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders.

    PubMed

    Portsteffen, H; Beyer, A; Becker, E; Epplen, C; Pawlak, A; Kunau, W H; Dodt, G

    1997-12-01

    Human peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous autosomal-recessive disease caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. These lethal diseases include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum's disease (IRD), three phenotypes now thought to represent a continuum of clinical features that are most severe in ZS, milder in NALD and least severe in IRD2. At least eleven PBD complementation groups have been identified by somatic-cell hybridization analysis compared to the eighteen PEX complementation groups that have been found in yeast. We have cloned the human PEX1 gene encoding a 147-kD member of the AAA protein family (ATPases associated with diverse cellular activities), which is the putative orthologue of Saccharomyces cerevisiae Pex1p (ScPex1p). Human PEX1 has been identified by computer-based 'homology probing' using the ScPex1p sequence to screen databases of expressed sequence tags (dbEST) for human cDNA clones. Expression of PEX1 rescued the cells from the biogenesis defect in human fibroblasts of complementation group 1 (CG1), the largest PBD complementation group. We show that PEX1 is mutated in CG1 patients. PMID:9398848

  14. Promotion of mitochondrial biogenesis by necdin protects neurons against mitochondrial insults

    PubMed Central

    Hasegawa, Koichi; Yasuda, Toru; Shiraishi, Chinatsu; Fujiwara, Kazushiro; Przedborski, Serge; Mochizuki, Hideki; Yoshikawa, Kazuaki

    2016-01-01

    Neurons rely heavily on mitochondria for their function and survival. Mitochondrial dysfunction contributes to the pathogenesis of neurodegenerative diseases such as Parkinson's disease. PGC-1α is a master regulator of mitochondrial biogenesis and function. Here we identify necdin as a potent PGC-1α stabilizer that promotes mitochondrial biogenesis via PGC-1α in mammalian neurons. Expression of genes encoding mitochondria-specific proteins decreases significantly in necdin-null cortical neurons, where mitochondrial function and expression of the PGC-1α protein are reduced. Necdin strongly stabilizes PGC-1α by inhibiting its ubiquitin-dependent degradation. Forced expression of necdin enhances mitochondrial function in primary cortical neurons and human SH-SY5Y neuroblastoma cells to prevent mitochondrial respiratory chain inhibitor-induced degeneration. Moreover, overexpression of necdin in the substantia nigra in vivo of adult mice protects dopaminergic neurons against degeneration in experimental Parkinson's disease. These data reveal that necdin promotes mitochondrial biogenesis through stabilization of endogenous PGC-1α to exert neuroprotection against mitochondrial insults. PMID:26971449

  15. The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

    PubMed Central

    Profant, Deborah A.; Roberts, Christopher J.; Koning, Ann J.; Wright, Robin L.

    1999-01-01

    In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain. PMID:10512876

  16. All-trans retinoic acid induces oxidative phosphorylation and mitochondria biogenesis in adipocytes[S

    PubMed Central

    Tourniaire, Franck; Musinovic, Hana; Gouranton, Erwan; Astier, Julien; Marcotorchino, Julie; Arreguin, Andrea; Bernot, Denis; Palou, Andreu; Bonet, M. Luisa; Ribot, Joan; Landrier, Jean-François

    2015-01-01

    A positive effect of all-trans retinoic acid (ATRA) on white adipose tissue (WAT) oxidative and thermogenic capacity has been described and linked to an in vivo fat-lowering effect of ATRA in mice. However, little is known about the effects of ATRA on mitochondria in white fat. Our objective has been to characterize the effect of ATRA on mitochondria biogenesis and oxidative phosphorylation (OXPHOS) capacity in mature white adipocytes. Transcriptome analysis, oxygraphy, analysis of mitochondrial DNA (mtDNA), and flow cytometry-based analysis of mitochondria density were performed in mature 3T3-L1 adipocytes after 24 h incubation with ATRA (2 µM) or vehicle. Selected genes linked to mitochondria biogenesis and function and mitochondria immunostaining were analyzed in WAT tissues of ATRA-treated as compared with vehicle-treated mice. ATRA upregulated the expression of a large set of genes linked to mtDNA replication and transcription, mitochondrial biogenesis, and OXPHOS in adipocytes, as indicated by transcriptome analysis. Oxygen consumption rate, mtDNA content, and staining of mitochondria were increased in the ATRA-treated adipocytes. Similar results were obtained in WAT depots of ATRA-treated mice. We conclude that ATRA impacts mitochondria in adipocytes, leading to increased OXPHOS capacity and mitochondrial content in these cells. PMID:25914170

  17. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients

    PubMed Central

    Gu, Bai-Wei; Apicella, Marisa; Mills, Jason; Fan, Jian-Meng; Reeves, Dara A.; French, Deborah; Podsakoff, Gregory M.; Bessler, Monica; Mason, Philip J.

    2015-01-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed “corrected” lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human

  18. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

    PubMed

    Gu, Bai-Wei; Apicella, Marisa; Mills, Jason; Fan, Jian-Meng; Reeves, Dara A; French, Deborah; Podsakoff, Gregory M; Bessler, Monica; Mason, Philip J

    2015-01-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human

  19. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    PubMed Central

    Giancaspero, Teresa A.; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M.; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD “chaperone.” The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

  20. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.

    PubMed

    Giancaspero, Teresa A; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

  1. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-06-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  2. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed Central

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  3. The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells.

    PubMed

    Qi, Hongying; Watanabe, Toshiaki; Ku, Hsueh-Yen; Liu, Na; Zhong, Mei; Lin, Haifan

    2011-02-01

    Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways. PMID:21106531

  4. Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of alpha-helical outer membrane proteins.

    PubMed

    Thornton, Nicolas; Stroud, David A; Milenkovic, Dusanka; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2010-02-26

    The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes. PMID:20026336

  5. Infectious Entry Pathway of Enterovirus B Species

    PubMed Central

    Marjomäki, Varpu; Turkki, Paula; Huttunen, Moona

    2015-01-01

    Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best “markers” of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication. PMID:26690201

  6. Similarities and differences in the biogenesis, processing and lysosomal targeting between zebrafish and human pro-Cathepsin D: functional implications.

    PubMed

    Follo, Carlo; Ozzano, Matteo; Montalenti, Claudia; Ekkapongpisit, Maneerat; Isidoro, Ciro

    2013-02-01

    The lysosomal protease Cathepsin D (CD) plays a role in neurodegenerative diseases, cancer, and embryo-fetus abnormalities. It is therefore of interest to know how this protein is synthesized in animal species used for modeling human diseases. Zebrafish (Danio rerio) is emerging as a valuable 'in vivo' vertebrate model for several human diseases. We have characterized the biogenetic pathways of zebrafish and human CD transgenically expressed in both human SH-SY5Y cells and zebrafish PAC2 cells. Differently from human CD, zebrafish CD was synthesized as a mono-glycosylated precursor (pro-CD) that was eventually processed into a single-chain mature polypeptide. In PAC2 cells, ammonium chloride and chloroquine impaired the N-glycosylation, and greatly stimulated the secretion, of pro-CD; still, a portion of un-glycosylated pro-CD reached the lysosomes and was processed to mature CD. The treatment with tunicamycin, which abrogates N-glycosylation, resulted in a similar effect. Zebrafish pro-CD was correctly processed when expressed in human cells, and its glycosylation, transport and maturation were not impaired by ammonium chloride. On the contrary, the transport and processing of human pro-CD expressed in zebrafish cells were profoundly altered: while the intermediate single-chain was not detectable, a small amount of double-chain mature CD still formed. This fact indicates that the enzyme machinery for single- to double-chain processing of mammal CD is present in zebrafish. Our data highlight the respective impact of the information imparted by the primary sequence and of the cellular transport and processing machineries in the biogenesis of lysosomal CD. PMID:23107604

  7. Biogenesis of the mitochondrial Tom40 channel in skeletal muscle from aged animals and its adaptability to chronic contractile activity.

    PubMed

    Joseph, Anna-Maria; Ljubicic, Vladimir; Adhihetty, Peter J; Hood, David A

    2010-06-01

    Evidence exists that mitochondrial content and/or function is reduced in muscle of aging individuals. The purposes of this study were to investigate the contribution of outer membrane protein import and assembly processes to this decline and to determine whether the assembly process could adapt to chronic contractile activity (CCA). Tom40 assembly into the translocases of the outer membrane (TOM complex) was measured in subsarcolemmal mitochondria obtained from young (6 mo old) and aged (36 mo old) Fischer 344 x Brown Norway animals. While the initial import of Tom40 did not differ between young and aged animals, its subsequent assembly into the final approximately 380 kDa complex was 2.2-fold higher (P < 0.05) in mitochondria from aged compared with young animals. This was associated with a higher abundance of Tom22, a protein vital for the assembly process. CCA induced a greater initial import and subsequent assembly of Tom40 in mitochondria from young animals, resulting in a CCA-induced 75% increase (P < 0.05) in Tom40 within mitochondria. This effect of CCA was attenuated in mitochondria from old animals. These data suggest that the import and assembly of proteins into the outer membrane do not contribute to reduced mitochondrial content or function in aged animals. Indeed, the greater assembly rate in mitochondria from aged animals may be a compensatory mechanism attempting to offset any decrements in mitochondrial content or function within aged muscle. Our data also indicate the potential of CCA to contribute to increased mitochondrial biogenesis in muscle through changes in the outer membrane import and assembly pathway. PMID:20107041

  8. Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

    PubMed Central

    Yamada, Hiroko; Horigome, Chihiro; Okada, Takafumi; Shirai, Chiharu; Mizuta, Keiko

    2007-01-01

    We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth. PMID:17804645

  9. IKKα-mediated biogenesis of miR-196a through interaction with Drosha regulates the sensitivity of cancer cells to radiotherapy.

    PubMed

    Fang, X; Jeong, J-H; Long, X; Park, S-J; Wang, D; Shuai, M; Wei, R; Li, C; Li, S; Zhang, S; Duran, M B; Lo, K-W; Tsao, S W; Glaser, R; Luo, Z; Feng, X; Tian, Y; Luo, J-L

    2016-09-01

    Radioresistance is a major obstacle in successful clinical cancer radiotherapy, and the underlying mechanisms are not clear. Here we show that IKKα-mediated miR-196a biogenesis via interaction with Drosha regulates the sensitivity of nasopharyngeal carcinoma (NPC) cells to radiotherapy. Phosphorylation of IKKα at T23 site (p-IKKαT23) promotes the binding of IKKα to Drosha that accelerates the processing of miR-196a primary transcripts, leading to increased expressions of both precursor and mature miR-196a. Dephosphorylation of p-IKKαT23 downregulates miR-196a expression and promotes the resistance of NPC cells to radiation treatment. The miR-196a mimic suppresses while its inhibitor promotes the resistance of NPC to radiation treatment. Importantly, the expression of p-IKKαT23 is positively related to the expression of miR-196a in human NPC tissues, and expression of p-IKKαT23 and miR-196a is inversely correlated with NPC clinical radioresistance. Thus, our studies establish a novel mechanistic link between the inactivation of IKKαT23-Drosha-miR-196a pathway and NPC radioresistance, and de-inactivation of IKKαT23-Drosha-miR-196a pathway would be an efficient way to restore the sensitivity of radioresistant NPC to radiotherapy. PMID:27058318

  10. eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference

    NASA Astrophysics Data System (ADS)

    Yi, Tingfang; Arthanari, Haribabu; Akabayov, Barak; Song, Huaidong; Papadopoulos, Evangelos; Qi, Hank H.; Jedrychowski, Mark; Güttler, Thomas; Guo, Cuicui; Luna, Rafael E.; Gygi, Steven P.; Huang, Stephen A.; Wagner, Gerhard

    2015-05-01

    MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.

  11. eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference

    PubMed Central

    Yi, Tingfang; Arthanari, Haribabu; Akabayov, Barak; Song, Huaidong; Papadopoulos, Evangelos; Qi, Hank H.; Jedrychowski, Mark; Güttler, Thomas; Guo, Cuicui; Luna, Rafael E.; Gygi, Steven P.; Huang, Stephen A.; Wagner, Gerhard

    2015-01-01

    MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID-domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centered RNA induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis. PMID:26018492

  12. Physical Exercise Regulates p53 Activity Targeting SCO2 and Increases Mitochondrial COX Biogenesis in Cardiac Muscle with Age

    PubMed Central

    Qi, Zhengtang; He, Jie; Su, Yuhui; He, Qiang; Liu, Jingxia; Yu, Lu; Al-Attas, Omar; Hussain, Tajamul; Ding, Shuzhe; Ji, Liu; Qian, Min

    2011-01-01

    The purpose of this study was to outline the timelines of mitochondrial function, oxidative stress and cytochrome c oxidase complex (COX) biogenesis in cardiac muscle with age, and to evaluate whether and how these age-related changes were attenuated by exercise. ICR/CD-1 mice were treated with pifithrin-μ (PFTμ), sacrificed and studied at different ages; ICR/CD-1 mice at younger or older ages were randomized to endurance treadmill running and sedentary conditions. The results showed that mRNA expression of p53 and its protein levels in mitochondria increased with age in cardiac muscle, accompanied by increased mitochondrial oxidative stress, reduced expression of COX subunits and assembly proteins, and decreased expression of most markers in mitochondrial biogenesis. Most of these age-related changes including p53 activity targeting cytochrome oxidase deficient homolog 2 (SCO2), p53 translocation to mitochondria and COX biogenesis were attenuated by exercise in older mice. PFTμ, an inhibitor blocking p53 translocation to mitochondria, increased COX biogenesis in older mice, but not in young mice. Our data suggest that physical exercise attenuates age-related changes in mitochondrial COX biogenesis and p53 activity targeting SCO2 and mitochondria, and thereby induces antisenescent and protective effects in cardiac muscle. PMID:21750704

  13. Physical exercise regulates p53 activity targeting SCO2 and increases mitochondrial COX biogenesis in cardiac muscle with age.

    PubMed

    Qi, Zhengtang; He, Jie; Su, Yuhui; He, Qiang; Liu, Jingxia; Yu, Lu; Al-Attas, Omar; Hussain, Tajamul; Ding, Shuzhe; Ji, Liu; Qian, Min

    2011-01-01

    The purpose of this study was to outline the timelines of mitochondrial function, oxidative stress and cytochrome c oxidase complex (COX) biogenesis in cardiac muscle with age, and to evaluate whether and how these age-related changes were attenuated by exercise. ICR/CD-1 mice were treated with pifithrin-μ (PFTμ), sacrificed and studied at different ages; ICR/CD-1 mice at younger or older ages were randomized to endurance treadmill running and sedentary conditions. The results showed that mRNA expression of p53 and its protein levels in mitochondria increased with age in cardiac muscle, accompanied by increased mitochondrial oxidative stress, reduced expression of COX subunits and assembly proteins, and decreased expression of most markers in mitochondrial biogenesis. Most of these age-related changes including p53 activity targeting cytochrome oxidase deficient homolog 2 (SCO2), p53 translocation to mitochondria and COX biogenesis were attenuated by exercise in older mice. PFTμ, an inhibitor blocking p53 translocation to mitochondria, increased COX biogenesis in older mice, but not in young mice. Our data suggest that physical exercise attenuates age-related changes in mitochondrial COX biogenesis and p53 activity targeting SCO2 and mitochondria, and thereby induces antisenescent and protective effects in cardiac muscle. PMID:21750704

  14. In vivo roles of BamA, BamB and BamD in the biogenesis of BamA, a core protein of the β-barrel assembly machine of Escherichia coli.

    PubMed

    Misra, Rajeev; Stikeleather, Ryan; Gabriele, Rebecca

    2015-03-13

    Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram-negative bacteria and in the mitochondria and chloroplasts of eukaryotes--two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP and, together with four outer membrane lipoproteins, BamBCDE, constitutes the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single-amino-acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F474L, it was demonstrated that the mutant BamA protein could not only assemble without BamD but also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data. PMID:24792419

  15. Disruption of snRNP biogenesis factors Tgs1 and pICln induces phenotypes that mirror aspects of SMN-Gemins complex perturbation in Drosophila, providing new insights into spinal muscular atrophy.

    PubMed

    Borg, Rebecca M; Fenech Salerno, Benji; Vassallo, Neville; Bordonne, Rémy; Cauchi, Ruben J

    2016-10-01

    The neuromuscular disorder, spinal muscular atrophy (SMA), results from insufficient levels of the survival motor neuron (SMN) protein. Together with Gemins 2-8 and Unrip, SMN forms the large macromolecular SMN-Gemins complex, which is known to be indispensable for chaperoning the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). It remains unclear whether disruption of this function is responsible for the selective neuromuscular degeneration in SMA. In the present study, we first show that loss of wmd, the Drosophila Unrip orthologue, has a negative impact on the motor system. However, due to lack of a functional relationship between wmd/Unrip and Gemin3, it is likely that Unrip joined the SMN-Gemins complex only recently in evolution. Second, we uncover that disruption of either Tgs1 or pICln, two cardinal players in snRNP biogenesis, results in viability and motor phenotypes that closely resemble those previously uncovered on loss of the constituent members of the SMN-Gemins complex. Interestingly, overexpression of both factors leads to motor dysfunction in Drosophila, a situation analogous to that of Gemin2. Toxicity is conserved in the yeast S. pombe where pICln overexpression induces a surplus of Sm proteins in the cytoplasm, indicating that a block in snRNP biogenesis is partly responsible for this phenotype. Importantly, we show a strong functional relationship and a physical interaction between Gemin3 and either Tgs1 or pICln. We propose that snRNP biogenesis is the pathway connecting the SMN-Gemins complex to a functional neuromuscular system, and its disturbance most likely leads to the motor dysfunction that is typical in SMA. PMID:27388936

  16. Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis

    PubMed Central

    Hurwitz, Stephanie N.; Conlon, Meghan M.; Rider, Mark A.; Brownstein, Naomi C.; Meckes, David G.

    2016-01-01

    Background Extracellular vesicles (EVs) are important mediators of cell-to-cell communication in healthy and pathological environments. Because EVs are present in a variety of biological fluids and contain molecular signatures of their cell or tissue of origin, they have great diagnostic and prognostic value. The ability of EVs to deliver biologically active proteins, RNAs and lipids to cells has generated interest in developing novel therapeutics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis and secretion remain unknown. Methods Here, we characterized vesicle secretion across the NCI-60 panel of human cancer cells by nanoparticle tracking analysis. Using CellMiner, the quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. Results Gene products positively associated with the quantity of exosomal-sized vesicles included vesicular trafficking classes of proteins with Rab GTPase function and sphingolipid metabolism. Positive correlates of larger microvesicle-sized vesicle secretion included gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. Conclusion These observations reveal new insights into genes involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. This study offers a foundation for further exploration of targets involved in EV biogenesis and secretion. PMID:27421995

  17. Thyroid Hormone Stimulation of Autophagy Is Essential for Mitochondrial Biogenesis and Activity in Skeletal Muscle.

    PubMed

    Lesmana, Ronny; Sinha, Rohit A; Singh, Brijesh K; Zhou, Jin; Ohba, Kenji; Wu, Yajun; Yau, Winifred W Y; Bay, Boon-Huat; Yen, Paul M

    2016-01-01

    Thyroid hormone (TH) and autophagy share similar functions in regulating skeletal muscle growth, regeneration, and differentiation. Although TH recently has been shown to increase autophagy in liver, the regulation and role of autophagy by this hormone in skeletal muscle is not known. Here, using both in vitro and in vivo models, we demonstrated that TH induces autophagy in a dose- and time-dependent manner in skeletal muscle. TH induction of autophagy involved reactive oxygen species (ROS) stimulation of 5'adenosine monophosphate-activated protein kinase (AMPK)-Mammalian target of rapamycin (mTOR)-Unc-51-like kinase 1 (Ulk1) signaling. TH also increased mRNA and protein expression of key autophagy genes, microtubule-associated protein light chain 3 (LC3), Sequestosome 1 (p62), and Ulk1, as well as genes that modulated autophagy and Forkhead box O (FOXO) 1/3a. TH increased mitochondrial protein synthesis and number as well as basal mitochondrial O2 consumption, ATP turnover, and maximal respiratory capacity. Surprisingly, mitochondrial activity and biogenesis were blunted when autophagy was blocked in muscle cells by Autophagy-related gene (Atg)5 short hairpin RNA (shRNA). Induction of ROS and 5'adenosine monophosphate-activated protein kinase (AMPK) by TH played a significant role in the up-regulation of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), the key regulator of mitochondrial synthesis. In summary, our findings showed that TH-mediated autophagy was essential for stimulation of mitochondrial biogenesis and activity in skeletal muscle. Moreover, autophagy and mitochondrial biogenesis were coupled in skeletal muscle via TH induction of mitochondrial activity and ROS generation. PMID:26562261

  18. Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis.

    PubMed

    Sui, Zhenhua; Nowak, Roberta B; Sanada, Chad; Halene, Stephanie; Krause, Diane S; Fowler, Velia M

    2015-07-23

    The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3(-/-) embryos, which are embryonic lethal by E18.5. Tmod3(-/-) embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3(-/-) fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3(-/-) MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3(-/-) MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3(-/-) MKs exhibit F-actin disturbances, and Tmod3(-/-) MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3(-/-) MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing. PMID:25964668

  19. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis

    PubMed Central

    Ipsaro, Jonathan J.; Haase, Astrid D.; Knott, Simon R.; Joshua-Tor, Leemor; Hannon, Gregory J.

    2012-01-01

    PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism that provides an essential protection for germ cell genomes against the activity of mobile genetic elements1. piRNA populations comprise a molecular definition of transposons that permits them to be distinguished from host genes and selectively silenced. piRNAs can be generated in two distinct ways. Primary piRNAs emanate from discrete genomic loci, termed piRNA clusters, and appear to be derived from long, single-stranded precursors2. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are likely formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner1,3. Secondary piRNAs arise during the adaptive ping-pong cycle, with their 5' termini being formed by the activity of PIWIs themselves2,4. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Zucchini, is a member of the phospholipase D family of phosphodiesterases, which includes both phospholipases and nucleases5–7. We have produced a dimeric, soluble fragment of the mouse Zucchini homolog (mZuc/PLD6) and have shown that it possesses single strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to PLD-family nucleases than to phospholipases. Considered together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs. PMID:23064227

  20. Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

    PubMed Central

    Hao, Enkui; Mukhopadhyay, Partha; Cao, Zongxian; Erdélyi, Katalin; Holovac, Eileen; Liaudet, Lucas; Lee, Wen-Shin; Haskó, György; Mechoulam, Raphael; Pacher, Pál

    2015-01-01

    Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX’s cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may

  1. BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex.

    PubMed

    Zhang, Lin; Duan, Zhikun; Zhang, Jiao; Peng, Lianwei

    2016-06-01

    Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3-CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase. PMID:27208269

  2. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis.

    PubMed

    Ipsaro, Jonathan J; Haase, Astrid D; Knott, Simon R; Joshua-Tor, Leemor; Hannon, Gregory J

    2012-11-01

    PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs. PMID:23064227

  3. Altered signaling for mitochondrial and myofibrillar biogenesis in skeletal muscles of patients with multiple sclerosis.

    PubMed

    Hansen, Dominique; Wens, Inez; Vandenabeele, Frank; Verboven, Kenneth; Eijnde, Bert O

    2015-07-01

    Patients with multiple sclerosis (pwMS) experience muscle weakness and lowered muscle oxidative capacity. To explore the etiology for the development of such muscle phenotype we studied skeletal muscle adenosine monophosphate (AMP)-activated protein kinase phosphorylation (phospho-AMPKα, governing mitochondrial biogenesis) and mammalian target of rapamycin phosphorylation (phospho-mTOR, governing myofibrillar biogenesis) in pwMS. After assessment of body composition, muscle strength, exercise tolerance, and muscle fiber type, muscle phospho-AMPKα and phospho-mTOR were assessed in 14 pwMS and 10 healthy controls (part 1). Next, an endurance exercise bout was executed by 9 pwMS and 7 healthy subjects, with assessment of changes in muscle phospho-AMPKα and phospho-mTOR (part 2). Increased basal muscle phospho-AMPKα and phospho-mTOR were present in MS (P < 0.01) and independently related to MS. Correlations between muscle phospho-AMPKα or phospho-mTOR and whole-body fat mass, peak oxygen uptake, and expanded disability status scale (P < 0.05) were found. After endurance exercise muscle phospho-AMPKα and phospho-mTOR remained increased in pwMS (P < 0.01). Muscle signaling cascades for mitochondrial and myofibrillar biogenesis are altered in MS and related to the impairment and disability level. These findings indicate a link between muscle signaling cascades and the level of disability and impairment, and thus may open a new area for the development of novel therapies for peripheral muscle impairment in MS. PMID:25666356

  4. Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

    PubMed Central

    Oxaran, Virginie; Ledue-Clier, Florence; Dieye, Yakhya; Herry, Jean-Marie; Péchoux, Christine; Meylheuc, Thierry; Briandet, Romain; Juillard, Vincent; Piard, Jean-Christophe

    2012-01-01

    The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used. PMID:23236417

  5. Turn up the power –pharmacological activation of mitochondrial biogenesis in mouse models

    PubMed Central

    Komen, J C; Thorburn, D R

    2014-01-01

    The oxidative phosphorylation (OXPHOS) system in mitochondria is responsible for the generation of the majority of cellular energy in the form of ATP. Patients with genetic OXPHOS disorders form the largest group of inborn errors of metabolism. Unfortunately, there is still a lack of efficient therapies for these disorders other than management of symptoms. Developing therapies has been complicated because, although the total group of OXPHOS patients is relatively large, there is enormous clinical and genetic heterogeneity within this patient population. Thus there has been a lot of interest in generating relevant mouse models for the different kinds of OXPHOS disorders. The most common treatment strategies tested in these mouse models have aimed to up-regulate mitochondrial biogenesis, in order to increase the residual OXPHOS activity present in affected animals and thereby to ameliorate the energy deficiency. Drugs such as bezafibrate, resveratrol and AICAR target the master regulator of mitochondrial biogenesis PGC-1α either directly or indirectly to manipulate mitochondrial metabolism. This review will summarize the outcome of preclinical treatment trials with these drugs in mouse models of OXPHOS disorders and discuss similar treatments in a number of mouse models of common diseases in which pathology is closely linked to mitochondrial dysfunction. In the majority of these studies the pharmacological activation of the PGC-1α axis shows true potential as therapy; however, other effects besides mitochondrial biogenesis may be contributing to this as well. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24102298

  6. Melatonin enhances mitophagy and mitochondrial biogenesis in rats with carbon tetrachloride-induced liver fibrosis.

    PubMed

    Kang, Jung-Woo; Hong, Jeong-Min; Lee, Sun-Mee

    2016-05-01

    Liver fibrosis leads to liver cirrhosis and failure, and no effective treatment is currently available. Growing evidence supports a link between mitochondrial dysfunction and liver fibrogenesis and mitochondrial quality control-based therapy has emerged as a new therapeutic target. We investigated the protective mechanisms of melatonin against mitochondrial dysfunction-involved liver fibrosis, focusing on mitophagy and mitochondrial biogenesis. Rats were treated with carbon tetrachloride (CCl4 ) dissolved in olive oil (0.5 mL/kg, twice a week, i.p.) for 8 wk. Melatonin was administered orally at 2.5, 5, and 10 mg/kg once a day. Chronic CCl4 exposure induced collagen deposition, hepatocellular damage, and oxidative stress, and melatonin attenuated these increases. Increases in mRNA and protein expression levels of transforming growth factor β1 and α-smooth muscle actin in response to CCl4 were attenuated by melatonin. Melatonin attenuated hallmarks of mitochondrial dysfunction, such as mitochondrial swelling and glutamate dehydrogenase release. Chronic CCl4 exposure impaired mitophagy and mitochondrial biogenesis, and melatonin attenuated this impairment, as indicated by increases in mitochondrial DNA and in protein levels of PTEN-induced putative kinase 1 (PINK1); Parkin; peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α); nuclear respiratory factor 1 (NRF1); and transcription factor A, mitochondrial (TFAM). CCl4 -mediated decreases in mitochondrial fission- and fusion-related proteins, such as dynamin-related protein 1 (DRP1) and mitofusin 2, were also attenuated by melatonin. Moreover, melatonin induced AMP-activated protein kinase (AMPK) phosphorylation. These results suggest that melatonin protects against liver fibrosis via upregulation of mitophagy and mitochondrial biogenesis, and may be useful as an anti-fibrotic treatment. PMID:26882442

  7. Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

    PubMed

    Whitney, Spencer M; Birch, Rosemary; Kelso, Celine; Beck, Jennifer L; Kapralov, Maxim V

    2015-03-17

    Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants. PMID:25733857

  8. Regulation of PGC-1α, a nodal regulator of mitochondrial biogenesis1234

    PubMed Central

    Fernandez-Marcos, Pablo J; Auwerx, Johan

    2011-01-01

    Mechanisms responsible for energy management in the cell and in the whole organism require a complex network of transcription factors and cofactors. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has emerged as a master regulator of mitochondrial biogenesis and function, thus becoming a crucial metabolic node. We present an overview of the mechanisms by which PGC-1α is regulated, including the transcriptional regulation of PGC-1α expression and the fine-tuning of its final activity via posttranslational modifications. PMID:21289221

  9. Straightforward synthesis of deuterated precursors to demonstrate the biogenesis of aromatic thiols in wine.

    PubMed

    Roland, Aurélie; Schneider, Rémi; Razungles, Alain; Le Guernevé, Christine; Cavelier, Florine

    2010-10-13

    Straightforward synthesis of labeled S-3-(hexan-1-ol)-glutathione and S-4-(4-methylpentan-2-one)-glutathione has been developed through a conjugate addition optimization study. Sauvignon blanc fermentation experiments with the [(2)H(10)] S-4-(4-methylpentan-2-one)-glutathione used as a tracer released the corresponding deuterated thiol, thus proving the direct relationship with the 4-mercapto-4-methylpentan-2-one under enological conditions. The conversion yield of such transformation was estimated to be close to 0.3%, opening an avenue for additional study on varietal thiol biogenesis. PMID:20825191

  10. Molecular Complexity Orchestrates Modulation of Phagosome Biogenesis and Escape to the Cytosol of macrophages by Francisella tularensis

    PubMed Central

    Asare, Rexford; Kwaik, Yousef Abu

    2010-01-01

    Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI-like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis subsp novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants co-localize persistently with the late endosomal/lysosomal marker LAMP-1 and fail to escape into the cytosol, as determined by fluorescence-based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro-environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis-mediated modulation of phagosome biogenesis, phagosomal escape, and bacterial proliferation within the cytosol is novel, complex, and involves

  11. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    SciTech Connect

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-07-18

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

  12. gigantea suppresses immutans variegation by interactions with cytokinin and gibberellin signaling pathways.

    PubMed

    Putarjunan, Aarthi; Rodermel, Steve

    2014-12-01

    The immutans (im) variegation mutant of Arabidopsis (Arabidopsis thaliana) is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in the control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GI, a central component of the circadian clock that plays a poorly understood role in diverse plant developmental processes. imgi2 mutants are late flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a development-specific derepression of cytokinin signaling that involves cross talk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, starch excess1 (sex1), perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GI and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis. PMID:25349324

  13. gigantea Suppresses immutans Variegation by Interactions with Cytokinin and Gibberellin Signaling Pathways1[W][OPEN

    PubMed Central

    Putarjunan, Aarthi; Rodermel, Steve

    2014-01-01

    The immutans (im) variegation mutant of Arabidopsis (Arabidopsis thaliana) is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in the control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GI, a central component of the circadian clock that plays a poorly understood role in diverse plant developmental processes. imgi2 mutants are late flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a development-specific derepression of cytokinin signaling that involves cross talk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, starch excess1 (sex1), perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GI and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis. PMID:25349324

  14. BUD22 Affects Ty1 Retrotransposition and Ribosome Biogenesis in Saccharomyces cerevisiae

    PubMed Central

    Dakshinamurthy, Arun; Nyswaner, Katherine M.; Farabaugh, Philip J.; Garfinkel, David J.

    2010-01-01

    A variety of cellular factors affect the movement of the retrovirus-like transposon Ty1. To identify genes involved in Ty1 virus-like particle (VLP) function, the level of the major capsid protein (Gag-p45) and its proteolytic precursor (Gag-p49p) was monitored in a subset of Ty1 cofactor mutants. Twenty-nine of 87 mutants contained alterations in the level of Gag; however, only bud22Δ showed a striking defect in Gag processing. BUD22 affected the +1 translational frameshifting event required to express the Pol proteins protease, integrase, and reverse transcriptase. Therefore, it is possible that the bud22Δ mutant may not produce enough functional Ty1 protease to completely process Gag-p49 to p45. Furthermore, BUD22 is required for 18S rRNA processing and 40S subunit biogenesis and influences polysome density. Together our results suggest that BUD22 is involved in a step in ribosome biogenesis that not only affects general translation, but also may alter the frameshifting efficiency of ribosomes, an event central to Ty1 retrotransposition. PMID:20498295

  15. Diacylglycerol Kinase α Regulates Tubular Recycling Endosome Biogenesis and Major Histocompatibility Complex Class I Recycling*

    PubMed Central

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2014-01-01

    Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. While a number of diacylglycerol kinase isoforms have been implicated in clathrin-dependent internalization, MHC I lacks the typical motifs known to mediate clathrin-dependent endocytosis. Here we show that depletion of diacylglycerol kinase α (DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of MHC I internalization. We demonstrate that DGKα knock-down causes accumulation of intracellular and surface MHC I, resulting from decreased degradation. Furthermore, we provide evidence that DGKα is required for the generation of phosphatidic acid required for tubular recycling endosome (TRE) biogenesis. Moreover, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with phosphatidic acid, we propose a positive feedback loop in which DGKα generates phosphatidic acid to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling. PMID:25248744

  16. GABP Transcription Factor (Nuclear Respiratory Factor 2) Is Required for Mitochondrial Biogenesis

    PubMed Central

    Yang, Zhong-Fa; Drumea, Karen; Mott, Stephanie; Wang, Junling

    2014-01-01

    Mitochondria are membrane-bound cytoplasmic organelles that serve as the major source of ATP production in eukaryotic cells. GABP (also known as nuclear respiratory factor 2) is a nuclear E26 transformation-specific transcription factor (ETS) that binds and activates mitochondrial genes that are required for electron transport and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding component of this transcription factor complex, from mouse embryonic fibroblasts (MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene expression. Gabpα loss modestly reduced mitochondrial mass, ATP production, oxygen consumption, and mitochondrial protein synthesis but did not alter mitochondrial morphology, membrane potential, apoptosis, or the expression of several genes that were previously reported to be GABP targets. However, the expression of Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for mitochondrial protein translation, was markedly reduced in Gabpα-null MEFs. We conclude that Gabp regulates Tfb1m expression and plays an essential, nonredundant role in mitochondrial biogenesis. PMID:24958105

  17. Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase.

    PubMed

    Shimada, Hiroshi; Mochizuki, Mariko; Ogura, Kan; Froehlich, John E; Osteryoung, Katherine W; Shirano, Yumiko; Shibata, Daisuke; Masuda, Shinji; Mori, Kazuki; Takamiya, Ken-Ichiro

    2007-10-01

    Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the albino cotyledon phenotype by complementation. CYO1 has a C(4)-type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons. PMID:17921316

  18. PGC-1α is Dispensable for Exercise-Induced Mitochondrial Biogenesis in Skeletal Muscle

    PubMed Central

    Rowe, Glenn C.; El-Khoury, Riyad; Patten, Ian S.; Rustin, Pierre; Arany, Zolt

    2012-01-01

    Exercise confers numerous health benefits, many of which are thought to stem from exercise-induced mitochondrial biogenesis (EIMB) in skeletal muscle. The transcriptional coactivator PGC-1α, a potent regulator of metabolism in numerous tissues, is widely believed to be required for EIMB. We show here that this is not the case. Mice engineered to lack PGC-1α specifically in skeletal muscle (Myo-PGC-1αKO mice) retained intact EIMB. The exercise capacity of these mice was comparable to littermate controls. Induction of metabolic genes after 2 weeks of in-cage voluntary wheel running was intact. Electron microscopy revealed no gross abnormalities in mitochondria, and the mitochondrial biogenic response to endurance exercise was as robust in Myo-PGC-1αKO mice as in wildtype mice. The induction of enzymatic activity of the electron transport chain by exercise was likewise unperturbed in Myo-PGC-1αKO mice. These data demonstrate that PGC-1α is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle, in sharp contrast to the prevalent assumption in the field. PMID:22848618

  19. Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation

    PubMed Central

    Cegelski, Lynette; Pinkner, Jerome S; Hammer, Neal D; Cusumano, Corinne K; Hung, Chia S; Chorell, Erik; Åberg, Veronica; Walker, Jennifer N; Seed, Patrick C; Almqvist, Fredrik; Chapman, Matthew R; Hultgren, Scott J

    2010-01-01

    Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1–dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds. PMID:19915538

  20. Temperature and Carbon Assimilation Regulate the Chlorosome Biogenesis in Green Sulfur Bacteria

    PubMed Central

    Tang, Joseph Kuo-Hsiang; Saikin, Semion K.; Pingali, Sai Venkatesh; Enriquez, Miriam M.; Huh, Joonsuk; Frank, Harry A.; Urban, Volker S.; Aspuru-Guzik, Alán

    2013-01-01

    Green photosynthetic bacteria adjust the structure and functionality of the chlorosome—the light-absorbing antenna complex—in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study, we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of C. tepidum grows slower and incorporates fewer BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays (a) smaller cross-sectional radius and overall size, (b) simplified BChl c homologs with smaller side chains, (c) blue-shifted Qy absorption maxima, and (d) a sigmoid-shaped circular dichroism spectra. Using a theoretical model, we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis. PMID:24047985

  1. Circular RNA biogenesis can proceed through an exon-containing lariat precursor

    PubMed Central

    Barrett, Steven P; Wang, Peter L; Salzman, Julia

    2015-01-01

    Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression programs, yet its function remains largely unknown. The presumed biogenesis of these RNAs involves a non-canonical ‘backsplicing’ event. Recent studies in mammalian cell culture posit that backsplicing is facilitated by inverted repeats flanking the circularized exon(s). Although such sequence elements are common in mammals, they are rare in lower eukaryotes, making current models insufficient to describe circularization. Through systematic splice site mutagenesis and the identification of splicing intermediates, we show that circular RNA in Schizosaccharomyces pombe is generated through an exon-containing lariat precursor. Furthermore, we have performed high-throughput and comprehensive mutagenesis of a circle-forming exon, which enabled us to discover a systematic effect of exon length on RNA circularization. Our results uncover a mechanism for circular RNA biogenesis that may account for circularization in genes that lack noticeable flanking intronic secondary structure. DOI: http://dx.doi.org/10.7554/eLife.07540.001 PMID:26057830

  2. β-Hydroxy-β-methylbutyrate, mitochondrial biogenesis, and skeletal muscle health.

    PubMed

    He, Xi; Duan, Yehui; Yao, Kang; Li, Fengna; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2016-03-01

    The metabolic roles of mitochondria go far beyond serving exclusively as the major producer of ATP in tissues and cells. Evidence has shown that mitochondria may function as a key regulator of skeletal muscle fiber types and overall well-being. Maintaining skeletal muscle mitochondrial content and function is important for sustaining health throughout the lifespan. Of great importance, β-hydroxy-β-methylbutyrate (HMB, a metabolite of L-leucine) has been proposed to enhance the protein deposition and efficiency of mitochondrial biogenesis in skeletal muscle, as well as muscle strength in both exercise and clinical settings. Specifically, dietary supplementation with HMB increases the gene expression of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), which represents an upstream inducer of genes of mitochondrial metabolism, coordinates the expression of both nuclear- and mitochondrion-encoded genes in mitochondrial biogenesis. Additionally, PGC-1α plays a key role in the transformation of skeletal muscle fiber type, leading to a shift toward type I muscle fibers that are rich in mitochondria and have a high capacity for oxidative metabolism. As a nitrogen-free metabolite, HMB holds great promise to improve skeletal muscle mass and function, as well as whole-body health and well-being of animals and humans. PMID:26573541

  3. Structure of BamA, an essential factor in outer membrane protein biogenesis.

    PubMed

    Albrecht, Reinhard; Schütz, Monika; Oberhettinger, Philipp; Faulstich, Michaela; Bermejo, Ivan; Rudel, Thomas; Diederichs, Kay; Zeth, Kornelius

    2014-06-01

    Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1-β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer. PMID:24914988

  4. Target-dependent biogenesis of cognate microRNAs in human cells.

    PubMed

    Bose, Mainak; Bhattacharyya, Suvendra N

    2016-01-01

    Extensive research has established how miRNAs regulate target mRNAs by translation repression and/or endonucleolytic degradation in metazoans. However, information related to the effect of target mRNA on biogenesis and stability of corresponding miRNAs in animals is limited. Here we report regulated biogenesis of cognate miRNAs by their target mRNAs. Enhanced pre-miRNA processing by AGO-associated DICER1 contributes to this increased miRNP formation. The processed miRNAs are loaded onto AGO2 to form functionally competent miRISCs both in vivo and also in a cell-free in vitro system. Thus, we identify an additional layer of posttranscriptional regulation that helps the cell to maintain requisite levels of mature forms of respective miRNAs by modulating their processing in a target-dependent manner, a process happening for miR-122 during stress reversal in human hepatic cells. PMID:27448149

  5. Mutants of Chlamydomonas: tools to study thylakoid membrane structure, function and biogenesis.

    PubMed

    de Vitry, C; Vallon, O

    1999-06-01

    The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and chloroplast biogenesis. C. reinhardtii has a photosynthesis apparatus similar to that of higher plants and it grows at rapid rate (generation time about 8 h). It is a facultative phototroph, which allows the isolation of mutants unable to perform photosynthesis and its sexual cycle allows a variety of genetic studies. Transformation of the nucleus and chloroplast genomes is easily performed. Gene transformation occurs mainly by homologous recombination in the chloroplast and heterologous recombination in the nucleus. Mutants are precious tools for studies of thylakoid membrane structure, photosynthetic function and assembly. Photosynthesis mutants affected in the biogenesis of a subunit of a protein complex usually lack the entire complex; this pleiotropic effect has been used in the identification of the other subunits, in the attribution of spectroscopic signals and also as a 'genetic cleaning' process which facilitates both protein complex purification, absorption spectroscopy studies or freeze-fracture analysis. The cytochrome b6f complex is not required for the growth of C. reinhardtii, unlike the case of photosynthetic prokaryotes in which the cytochrome complex is also part of the respiratory chain, and can be uniquely studied in Chlamydomonas by genetic approaches. We describe in greater detail the use of Chlamydomonas mutants in the study of this complex. PMID:10433117

  6. Biogenesis and Early Life on Earth and Europa: Favored by an Alkaline Ocean?

    NASA Astrophysics Data System (ADS)

    Kempe, Stephan; Kazmierczak, Jozef

    2002-03-01

    Recent discoveries about Europa - the probable existence of a sizeable ocean below its ice crust; the detection of hydrated sodium carbonates, among other salts; and the calculation of a net loss of sodium from the subsurface - suggest the existence of an alkaline ocean. Alkaline oceans (nicknamed "soda oceans" in analogy to terrestrial soda lakes) have been hypothesized also for early Earth and Mars on the basis of mass balance considerations involving total amounts of acids available for weathering and the composition of the early crust. Such an environment could be favorable to biogenesis since it may have provided for very low Ca2+ concentrations mandatory for the biochemical function of proteins. A rapid loss of CO2 from Europa's atmosphere may have led to freezing oceans. Alkaline brine bubbles embedded in ice in freezing and impact-thawing oceans could have provided a suitable environment for protocell formation and the large number of trials needed for biogenesis. Understanding these processes could be central to assessing the probability of life on Europa.

  7. Mining the surface proteome of tomato (Solanum lycopersicum) fruit for proteins associated with cuticle biogenesis

    PubMed Central

    Yeats, Trevor H.; Howe, Kevin J.; Matas, Antonio J.; Buda, Gregory J.; Thannhauser, Theodore W.; Rose, Jocelyn K. C.

    2010-01-01

    The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the ∼200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed. PMID:20571035

  8. Eukaryotic Initiation Factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

    SciTech Connect

    Guo, Jianjun; Jin, Zhaoqing; Yang, Xiaohan; Li, Jian-Feng; Chen, Jay

    2011-01-01

    We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

  9. Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

    PubMed Central

    Machado-Pinilla, Rosario; Liger, Dominique; Leulliot, Nicolas; Meier, U. Thomas

    2012-01-01

    The AAA+ ATPases pontin and reptin function in a staggering array of cellular processes including chromatin remodeling, transcriptional regulation, DNA damage repair, and assembly of macromolecular complexes, such as RNA polymerase II and small nucleolar (sno) RNPs. However, the molecular mechanism for all of these AAA+ ATPase associated activities is unknown. Here we document that, during the biogenesis of H/ACA RNPs (including telomerase), the assembly factor SHQ1 holds the pseudouridine synthase NAP57/dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry NAP57 from SHQ1. Significantly, the NAP57 domain captured by SHQ1 harbors most mutations underlying X-linked dyskeratosis congenita (X-DC) implicating the interface between the two proteins as a target of this bone marrow failure syndrome. Homing in on the essential first steps of H/ACA RNP biogenesis, our findings provide the first insight into the mechanism of action of pontin and reptin in the assembly of macromolecular complexes. PMID:22923768

  10. Flavan-3-ol fraction from cocoa powder promotes mitochondrial biogenesis in skeletal muscle in mice

    PubMed Central

    2014-01-01

    Background Numerous clinical studies have reported that ingestion of chocolate has reduced risk of metabolic syndrome. In order to elucidate the mechanism, we evaluated the influence of flavan-3-ols derived from cocoa powder on energy metabolism in mice using an indirect calorimetric method. Method The mice were divided into two groups, and administered either distilled water or 50 mg/kg of flavan-3-ol fraction for 2 weeks. At the end of the experimental period, animals were sacrificed after blood pressure and the mean respiratory exchange ratio (RER) over 24 hours were measured. Results The mean respiratory exchange ratio (RER) over 24 hours was reduced significantly in the flavan-3-ols group. The mean blood pressure was significantly decreased in flavan-3-ols treatment group compared with control group. The protein level of carnitine palmitoyltransferase 2 (CPT2) was increased significantly by flavan-3-ols in skeletal muscle, but not in liver. Uncoupling protein (UCP) 1 was increased significantly in brown adipose tissue by flavan-3-ols. The mitochondria copy number in gastrocnemius and soleus muscles and brown adipose tissue were increased significantly by administration of flavan-3-ol fraction. Conclusion These results suggest that flavan-3-ols enhances lipolysis and promotes mitochondrial biogenesis. We conclude that improvement of metabolic syndrome risk factors following ingestion of chocolate may be induced, in part, by the mitochondrial biogenesis-promoting effect of flavan-3-ols. PMID:24708519

  11. Identification and Expression Analysis of Ribosome Biogenesis Factor Co-orthologs in Solanum lycopersicum

    PubMed Central

    Simm, Stefan; Fragkostefanakis, Sotirios; Paul, Puneet; Keller, Mario; Einloft, Jens; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-01-01

    Ribosome biogenesis involves a large inventory of proteinaceous and RNA cofactors. More than 250 ribosome biogenesis factors (RBFs) have been described in yeast. These factors are involved in multiple aspects like rRNA processing, folding, and modification as well as in ribosomal protein (RP) assembly. Considering the importance of RBFs for particular developmental processes, we examined the complexity of RBF and RP (co-)orthologs by bioinformatic assignment in 14 different plant species and expression profiling in the model crop Solanum lycopersicum. Assigning (co-)orthologs to each RBF revealed that at least 25% of all predicted RBFs are encoded by more than one gene. At first we realized that the occurrence of multiple RBF co-orthologs is not globally correlated to the existence of multiple RP co-orthologs. The transcript abundance of genes coding for predicted RBFs and RPs in leaves and anthers of S. lycopersicum was determined by next generation sequencing (NGS). In combination with existing expression profiles, we can conclude that co-orthologs of RBFs by large account for a preferential function in different tissue or at distinct developmental stages. This notion is supported by the differential expression of selected RBFs during male gametophyte development. In addition, co-regulated clusters of RBF and RP coding genes have been observed. The relevance of these results is discussed. PMID:25698879

  12. Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis.

    PubMed

    Kobayashi, Koichi; Kondo, Maki; Fukuda, Hiroaki; Nishimura, Mikio; Ohta, Hiroyuki

    2007-10-23

    The biogenesis of thylakoid membranes, an indispensable event for the photoautotrophic growth of plants, requires a significant increase in the level of the unique thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG), which constitutes the bulk of membrane lipids in chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. Here we report the identification and characterization of an Arabidopsis mutant showing a complete defect in MGDG synthase 1. The mutant seeds germinated as small albinos only in the presence of sucrose. The seedlings lacked galactolipids and had disrupted photosynthetic membranes, leading to the complete impairment of photosynthetic ability and photoautotrophic growth. Moreover, invagination of the inner envelope, which is not seen in mature WT chloroplasts, was observed in the mutant, supporting an old hypothesis that envelope invagination is a major event in early chloroplast biogenesis. In addition to the defective seedling phenotype, embryo development was arrested in the mutant, although seeds with impaired embryos could germinate heterotrophically. These results demonstrate the importance of galactolipids not only in photosynthetic growth but also in embryogenesis. PMID:17940034

  13. ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.

    PubMed

    Zipeto, Maria Anna; Court, Angela C; Sadarangani, Anil; Delos Santos, Nathaniel P; Balaian, Larisa; Chun, Hye-Jung; Pineda, Gabriel; Morris, Sheldon R; Mason, Cayla N; Geron, Ifat; Barrett, Christian; Goff, Daniel J; Wall, Russell; Pellecchia, Maurizio; Minden, Mark; Frazer, Kelly A; Marra, Marco A; Crews, Leslie A; Jiang, Qingfei; Jamieson, Catriona H M

    2016-08-01

    Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling. PMID:27292188

  14. Target-dependent biogenesis of cognate microRNAs in human cells

    PubMed Central

    Bose, Mainak; Bhattacharyya, Suvendra N.

    2016-01-01

    Extensive research has established how miRNAs regulate target mRNAs by translation repression and/or endonucleolytic degradation in metazoans. However, information related to the effect of target mRNA on biogenesis and stability of corresponding miRNAs in animals is limited. Here we report regulated biogenesis of cognate miRNAs by their target mRNAs. Enhanced pre-miRNA processing by AGO-associated DICER1 contributes to this increased miRNP formation. The processed miRNAs are loaded onto AGO2 to form functionally competent miRISCs both in vivo and also in a cell-free in vitro system. Thus, we identify an additional layer of posttranscriptional regulation that helps the cell to maintain requisite levels of mature forms of respective miRNAs by modulating their processing in a target-dependent manner, a process happening for miR-122 during stress reversal in human hepatic cells. PMID:27448149

  15. Mutant p53 inhibits miRNA biogenesis by interfering with the microprocessor complex.

    PubMed

    Garibaldi, F; Falcone, E; Trisciuoglio, D; Colombo, T; Lisek, K; Walerych, D; Del Sal, G; Paci, P; Bossi, G; Piaggio, G; Gurtner, A

    2016-07-21

    Downregulation of microRNAs (miRNAs) is commonly observed in cancers and promotes tumorigenesis suggesting that miRNAs may function as tumor suppressors. However, the mechanism through which miRNAs are regulated in cancer, and the connection between oncogenes and miRNA biogenesis remain poorly understood. The TP53 tumor-suppressor gene is mutated in half of human cancers resulting in an oncogene with gain-of-function activities. Here we demonstrate that mutant p53 (mutp53) oncoproteins modulate the biogenesis of a subset of miRNAs in cancer cells inhibiting their post-transcriptional maturation. Interestingly, among these miRNAs several are also downregulated in human tumors. By confocal, co-immunoprecipitation and RNA-chromatin immunoprecipitation experiments, we show that endogenous mutp53 binds and sequesters RNA helicases p72/82 from the microprocessor complex, interfering with Drosha-pri-miRNAs association. In agreement with this, the overexpression of p72 leads to an increase of mature miRNAs levels. Moreover, functional experiments demonstrate the oncosuppressive role of mutp53-dependent miRNAs (miR-517a, -519a, -218, -105). Our study highlights a previously undescribed mechanism by which mutp53 interferes with Drosha-p72/82 association leading, at least in part, to miRNA deregulation observed in cancer. PMID:26996669

  16. Cytochromes c biogenesis in a photosynthetic bacterium requires a periplasmic thioredoxin-like protein.

    PubMed Central

    Beckman, D L; Kranz, R G

    1993-01-01

    Rhodobacter capsulatus is a Gram-negative photosynthetic bacterium that requires c-type cytochromes for photosynthetic electron transport. Our studies demonstrate that the gene helX is required for the biogenesis of c-type cytochromes in R. capsulatus. A helX chromosomal deletion mutant cannot grow photosynthetically, due to a deficiency of all c-type cytochromes. The predicted amino acid sequence of the helX gene product (176 residues) is related to that of thioredoxin and shares active-site homology with protein disulfide isomerase. Cytochrome c2-alkaline phosphatase gene fusions are used to show that HelX is not required for the transcription, translation, or secretion of apocytochrome c2. HelX-alkaline phosphatase and HelX-beta-galactosidase gene fusions are used to demonstrate that HelX is a periplasmic protein, which is consistent with the presence of a typical signal sequence in HelX. Based on these results, we propose HelX functions as a periplasmic disulfide oxidoreductase that is essential for cytochromes c biogenesis. This role is in accordance with the observation that both heme and the cysteines of apocytochromes c (Cys-Xaa-Yaa-Cys-His) must be in the reduced state for covalent linkage between the two moieties to occur. PMID:8384715

  17. Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis